Western Blotting, ELISA and Cell Imaging

Transcription

1 Western Blotting, ELISA and Cell Imaging Western blotting, ELISA and cellular imaging methods share many of the same principles and require many of the same reagents and procedural steps. In each case, protein samples are adsorbed or fixed to a surface (e.g., membrane, polystyrene microplate) and then probed with an antibody or other target-specific binding molecule. Through a reporter enzyme or other tag that is attached to the primary or secondary probe, the presence of target analyte is made visible as a measurable color or light-emitting signal. Reagents such as secondary antibodies, and washing and blocking buffers, are common to all three detection methods. Surface materials, assay substrates and other components are specific to individual methods. We offer a complete line of reagents and kits for both traditional and state-of-the-art varieties of these essential protein imaging methods. 127

2 Thermo Scientific Western Blotting, ELISA and Cell Imaging Products Secondary Probes for Detection Methods Secondary Antibodies 129 IP Detection Reagents 133 DyLight Dye and other Fluor-Conjugates 134 Anti-Label and Anti-Tag Antibodies 137 His Tag and Phosphoprotein Probes 138 Immunoglobulins and Sera 139 Antibody-Binding Proteins (Protein A, G, L) 140 Biotin-Binding Proteins (Avidin, Streptavidin, etc.) 141 Western Blotting Reagents Membranes for Blotting 145 Protein Stain for Membranes 14 Antibody Extender Solution 14 Signal Enhancer 147 Blocking Buffers 148 Wash and Transfer Buffers 152 Chemiluminescent Substrates 153 Fluorescent Western Blotting Kits 158 In-Gel Detection Kits 10 X-ray Film and Accessories 11 Stripping Buffers 13 Colorimetric Substrates 15 ELISA Reagents and Consumables Polystyrene Plates and Other Consumables 18 Coated Microplates 170 Blocking Buffers and Wash Buffers 179 Choosing a Detection Method 180 Chemiluminescent Substrates 181 Fluorogenic Substrates 183 Colorimetric Substrates 184 Immunohistochemistry Reagents Blocking Serum and Fixative 187 Staining Kits and Substrates 188 Cell Imaging Technologies 191 Nucleic Acid Labeling and Detection Reagents Gel Shift (LightShift) Kit 192 DNA Labeling Kits 193 Northern and Southern Blotting Kits 194 Detection Module, Luciferin 195 Molecular Biology Reagents

3 Secondary Antibodies Affinity-Purified Secondary Antibodies Definition and Benefits of Secondary Antibodies Western blotting, ELISA, immunohistochemistry and other imaging methods typically involve the use of an unlabeled, specific antibody as the initial probe for the target analyte. Although its specificity is the basis for analyte detection, this unlabeled primary antibody is not directly visible to an imaging system. A second probing step is required one involving an antibody that binds to the specific species and class of primary antibody immunoglobulin and is also conjugated to a detectable tag (reporter enzyme or fluorophore). Because of their common use as secondary probes, all antibodies that are specific to other immunoglobulins are called secondary antibodies. The availability of secondary antibodies that are specific for many different immunoglobulin species, classes, subclasses and fragments greatly facilitates the design and execution of specific assays. Primary antibodies, because they are usually produced as monoclonal immunoglobulins, are typically more expensive and not readily available; using secondary antibodies avoids the challenges and costs of labeling or procuring pre-labeled primary antibodies. Using a secondary probing step usually provides signal amplification for the assay because more than one secondary antibody molecule can bind to each primary antibody molecule. Finally, in certain multiplex applications, a single secondary antibody can provide for simultaneous detection of two primary antibodies. Thermo Scientific Pierce Secondary Antibodies are affinity-purified polyclonal antibodies with well-characterized specificity for particular target immunoglobulins. The diversity of options with regard to species and conjugate types provides for most assay systems in proteomics research. Unconjugated secondary antibodies provide for customized labeling needs. For a complete listing of Thermo Scientific DyLight Dye, fluorescein and other fluor-conjugated secondary antibodies, see pages Thermo Scientific Secondary Detection Reagents Pierce Secondary Antibodies Affinity-purified, polyclonal antibodies for a variety of applications. Thermo Scientific Pierce Secondary Antibodies are affinity-purified polyclonal antibodies with well-characterized specificity for particular target immunoglobulins. As polyclonals, they are relatively easy to produce in abundance, which translates to their low cost. Each antibody is affinity-purified using its target antigen (i.e., the target primary antibody species, class, subclass and fragment) coupled to agarose resin. Affinity purification eliminates nonspecific antibodies, resulting in high specificity and low background. The purification process involves an elution procedure, which yields antibodies with high avidity. The antibodies exhibit maximal binding to antigen and minimal cross-reactivity to other molecules. Conjugated antibodies are affinity-purified before being labeled and contain bovine serum albumin as a stabilizer. Appropriate working concentrations for secondary antibodies depend upon the assay method, enzyme and substrate system, and imaging instrumentation to be used. Empirical testing is required for optimization. General guidelines for dilution of 1 mg/ml secondary antibody stock solutions for typical applications are provided in the accompanying table. Considerations for Choosing a Secondary Antibody Species and Ig specificity Choose a secondary antibody specific for the species and immunoglobulin class of the primary antibody. For example, if the primary antibody is a monoclonal mouse IgG, then choose an anti-mouse IgG secondary antibody. Conjugate type Choose a secondary antibody that is conjugated with biotin, peroxidase (HRP), alkaline phosphatase (AP) or a fluorophore, based on the desired tertiary probing step or substrate-detection method to be used. Choose an unconjugated secondary antibody only if a tertiary antibody will be used or another type of custom labeling step is required. Fragment and chain specificity Except for special situations, choose a secondary antibody that binds both heavy and light chains (H+L) of the target immunoglobulin. This provides maximum assay sensitivity by allowing multiple secondary antibodies to bind to each primary antibody. Minimized cross-reactivity Choose a pre-adsorbed (min x Sr Prot) secondary antibody if the sample and assay system includes components of serum from a species that might otherwise cross-react with the secondary antibody. For example, if a Western blot sample was mouse serum, then choose a secondary antibody identified as (min x Ms Sr Prot). Host species source Choose a secondary antibody from a host (source) that is not a target of other secondary antibodies that might be used in a multplex assay. Source fragment composition Most secondary antibodies are composed of polyclonal, whole IgG molecules purified from the host species. Selected antibodies are available as specific fragments (e.g., F(ab') 2 ) of the host immunoglobulins. Choose these specialized secondary antibody preparations when the assay system necessitates the avoidance of certain fragments (e.g., Fc). Suggested dilution ranges in typical applications for secondary antibodies (from 1 mg/ml stock). Conjugate ELISA Western Blotting Immunohistochemistry Alkaline 1:5,000-1:50,000 1:2,500-1:25,000 1:500-1:5,000 Phosphatase Peroxidase 1:5,000-1:200,000 1:25,000-1:500,000 1:500-1:5,000 (for Thermo Scientific SuperSignal ELISA Products) (for Thermo Scientific SuperSignal West Products) Fluorescein 1:50-1:200 Rhodamine 1:50-1:200 Additional dry ice and/or freight charges Additional hazardous handling charge *Trademark, see Trademark Index To order or for technical support, contact your local office or distributor. See pages -2,

4 Secondary Antibodies Thermo Scientific Pierce Secondary Antibodies are supplied lyophilized or in solution and are stable for at least 1 year when stored as directed. Lyophilized antibodies are formatted to provide a buffered and stabilized solution when reconstituted in water at approximately 1 mg/ml (individual product inserts specify lot-specific values). Antibodies provided in solution are also stabilized and buffered at approximately 1 mg/ml. Storage of stock solutions in 50% ethylene glycol or frozen in single-use aliquots can greatly extend the shelf-life of secondary antibodies. (See page 311 for additional information about these accessory products.) Selected secondary antibodies have been further purified to minimize cross-reactivities to serum proteins of other species. This purification is accomplished by adsorbing the antigen-purified secondary antibody sample to a secondary affinity column containing immobilized serum proteins of selected species. In the following product tables, these antibody products that have been pre-adsorbed to species serum proteins are indicated by the code min x Sp Sr Prot, where particular species are specified according to the following Species Key: Key to species abbreviations. Bv = Bovine Gu = Guinea Pig Hs = Horse Rt = Rat Ch = Chicken Ha = Hamster Ms = Mouse Sh = Sheep Gt = Goat Hn = Human Rb = Rabbit Sw = Swine Product # and Package Size Description Specificity Source Unconjugated Biotin Peroxidase Alk. Phos. Fluorophore Anti-CHICKEN Chicken IgY (H+L) Rabbit See page mg 1.5 ml 1.5 ml Anti-GOAT Goat IgG (H+L) Mouse See page 135 (min x HnMsRb Sr Prot) 1.5 mg 1 ml 1 ml Goat IgG (H+L) Rabbit See page mg 1.5 mg 1.5 ml 1 ml Goat IgG [F(ab ) 2 ] Rabbit See page ml 1.5 ml Goat IgG (Fc) Rabbit See page mg 1.5 ml 1 ml Goat IgG (H+L) Rabbit (min x Hn Sr Prot) F(ab ) mg 0.5 ml Anti-HAMSTER Hamster IgG (H+L) Goat mg 1.5 mg Anti-HORSE Horse IgG (H+L) Goat mg Anti-HUMAN Human IgG (H+L) Goat See page 13 2 mg 1.5 mg 2 ml 1 ml Human IgG Goat Gamma Chain Specific 0.5 mg Human IgG (H+L) Goat See page 13 (min x BvHsMs Sr Prot) 1.5 mg 1.5 ml 1.5 ml Human IgG [F(ab ) 2 ] Goat See page 13 2 mg 2 ml 1 ml Human IgG [F(ab ) 2 ] Goat (min x BvHsMs Sr Prot) 1.5 mg 1.5 ml Human IgG (Fc) Goat (min x BvHsMs Sr Prot) 1 mg 1 ml Human IgM (Fc5µ) Goat See page 13 2 mg 2 ml Human IgM (µ) Goat mg 0.5 mg Human IgA (α) Goat See page 13 2 mg 2 ml 1 ml Human IgA + IgG Goat IgM (H+L) 2 mg 2 ml 2 ml 1 ml Human Kappa Chain Goat mg 0.5 mg Human Lambda Chain Goat mg Human IgG (H+L) Mouse (min x Ms Sr Prot) 2 mg 1.5 ml Human IgG (H+L) Mouse (min x BvHsMs Sr Prot) 1.5 mg 1 ml See Table at the top of this page for the Key to Abbreviations. 130 To order or for technical support, contact your local office or distributor. See pages -2, 3.

5 Secondary Antibodies Product # and Package Size Description Specificity Source Unconjugated Biotin Peroxidase Alk. Phos. Fluorophore Anti-HUMAN Human IgG (H+L) Rabbit continued 2 mg 1.5 ml Human IgG (Fc) Rabbit See page 13 2 mg 1.5 ml 1.5 ml 1 ml Human IgG (Fc) Goat 3113 F(ab ) 2 1 mg Human IgG (H+L) Goat 3115 F(ab ) 2 1 mg Human IgA + IgG + Goat See page 13 IgM (H+L) F(ab ) 2 Anti-MOUSE Mouse IgA (α) Goat 3119 (min x Hn Sr Prot) 1 mg Mouse IgA + IgG + IgM (H+L) Goat mg Mouse IgG (H+L) Goat See page 13 2 mg 2 ml 2 ml 1 ml Mouse IgG (H+L) Goat See page 13 (min x BvHnHs Sr Prot) 1.5 mg 1.5 mg 1.5 ml 1 ml Mouse IgG [F(ab ) 2 ] Goat See page 13 2 mg 2 ml 2 ml 1 ml Mouse IgG (Fc) Goat See page 13 2 mg 2 ml 2 ml 1 ml Mouse IgG (Fc) Goat (min x BvHnHs Sr Prot) 1.5 mg 1.5 ml 1 ml Mouse IgM (µ) Goat See page 13 2 mg 0.5 mg 2 ml 1 ml Mouse IgG + IgM Goat (H+L) 2 mg 2 ml 2 ml 1 ml Mouse IgG + IgM (H+L) Goat (min x BvHnHs Sr Prot) 1.5 ml 1 ml Mouse IgG (Fcγ) Goat See page 13 (subclasses 1+2a+2b+3) 1 mg (min x BvHnRb Sr Prot) Mouse IgG (Fcγ) Goat 3123 See page 13 subclass 1 specific 1 mg (min x BvHnRb Sr Prot) Mouse IgG (Fcγ) Goat See page 13 subclass 2a specific 1 mg (min x BvHnRb Sr Prot) Mouse IgG (H+L) Horse mg 1.5 mg Mouse IgG (H+L) Rabbit See page 13 2 mg 1.5 ml 1 ml Mouse IgG (H+L) Rabbit (min x Hn Sr Prot) 1.5 mg 1 ml 0.5 ml Mouse IgG [F(ab ) 2 ] Rabbit See page 13 2 mg 1.5 ml 1 ml Mouse IgG (Fc) Rabbit See page 13 2 mg 1.5 ml 1.5 ml 1 ml Mouse IgM (µ) Rabbit See page 13 2 mg 1.5 ml 1 ml Mouse IgG + IgM Rabbit (H+L) 2 mg 1.5 ml 1 ml Mouse IgG (H+L) Goat See page 13 (min x BvHnHs Sr Prot) F(ab ) 2 1 mg 0.5 ml Mouse IgM (µ) Goat F(ab ) 2 1 mg Mouse IgM (µ) Goat 3118 (min x BvHnHs Sr Prot) F(ab ) 2 1 mg Mouse IgG + IgM (H+L) Goat (min x BvHnHs Sr Prot) F(ab ) ml See Table at the top of page 130 for the Key to Abbreviations. Stabilized, pre-diluted format also available; see next page. Additional dry ice and/or freight charges Additional hazardous handling charge *Trademark, see Trademark Index To order or for technical support, contact your local office or distributor. See pages -2,

6 Secondary Antibodies Product # and Package Size Description Specificity Source Unconjugated Biotin Peroxidase Alk. Phos. Fluorophore Anti-RABBIT Rabbit IgG (H+L) Donkey See page 13 (min x BvChGtGuHaHn 1 mg 0.5 ml 0.5 ml 0.5 ml HsMsRtSh Sr Prot) Rabbit IgG (H+L) Goat See page 13 2 mg 1.5 mg 2 ml 1 ml Rabbit IgG (H+L) Goat See page 13 (min x Hn Sr Prot) 1.5 mg 1.5 ml 1.5 ml 1 ml Rabbit IgG [F(ab ) 2 ] Goat See page 13 2 mg 2 ml 2 ml 1 ml Rabbit IgG (Fc) Goat mg 2 ml 1 ml Rabbit IgG (H+L) Mouse See page 13 (min x GtHnMsSh Sr Prot) 1.5 mg 1 ml 1 ml Rabbit IgG (H+L) Goat See page 13 (min x HnMsRt Sr Prot) F(ab ) 2 1 mg Anti-RAT Rat IgG (H+L) Goat See page 13 2 mg 2 ml 2 ml 1 ml Rat IgG [F(ab ) 2 ] Goat ml Rat IgG (Fc) Goat See page 13 2 mg 2 ml Rat IgM (µ) Goat mg 2 ml 2 ml Rat IgG (H+L) Rabbit mg 1.5 mg Rat IgG (H+L) Rabbit (min x Ms Sr Prot) 0.5 mg Anti-SHEEP Sheep IgG (H+L) Rabbit See page 13 2 mg 1.5 mg 1.5 ml 1 ml See Table at the top of page 130 for the Key to Abbreviations. Stabilized, pre-diluted format also available; see below. Stabilized HRP Conjugates Pre-diluted, stable solutions of our most popular secondary antibodies. Stabilized HRP Conjugates are secondary antibody conjugates with horseradish peroxidase (HRP) enzyme that are stabilized in pre-diluted form for greater accuracy and convenience in preparing working solutions. Thermo Scientific Stabilized HRP Conjugates are accurately prepared, dispensed and supplied at 10 µg/ml (100X more dilute than typical 1 mg/ml preparations), eliminating the inaccuracies associated with two-stage dilution schemes required with traditional conjugate preparations for use with chemiluminescent substrates and other high-sensitivity detection methods. The liquid formulation of each prediluted HRP-conjugate is stable at 4 C, eliminating the need to freeze stock solutions for storage Stabilized Goat Anti-Mouse IgG (H+L), Peroxidase Conjugated 3240 Stabilized Goat Anti-Rabbit IgG (H+L), Peroxidase Conjugated 2 ml 2 ml Antibody Stabilizers and Storage Solutions Polyclonal secondary antibodies are typically stable when stored frozen as concentrated stock solutions in simple phosphate or Tris buffers containing sodium azide or other antimicrobial agents. Most uses of secondary antibodies require more than 1,000-fold dilution using only a few microliters of stock, and the daily need for Western blotting or ELISA experiments inevitably leads to repeated freeze-thaw cycles that are damaging to the antibody and conjugated enzyme. Freeze-thaw cycles can be avoided by storing a concentrated antibody stock as a mixture containing 20-50% of an anti-freeze compound such as glycerol or ethylene glycol. Glycerol is most frequently used for this purpose but commonly contains impurities that can adversely affect protein function. High-quality ethylene glycol is superior to glycerol for this purpose. Thermo Scientific Pierce Peroxidase Stabilizer is an antifreeze solution that provides the highest purity and performance for antibodies conjugated with horseradish peroxidase (HRP). The most convenient option is to store antibodies at the working concentration so that dilutions do not have to be repeated for each use. This is rarely possible with typical buffers, but Thermo Scientific Guardian Peroxidase Conjugate Stabilizer/Diluent provides this sort of protection for antibodies conjugated with horseradish peroxidase. Antibodies can be stored at 1-1,000 ng/ml for more than six months at room temperature without losing activity. For pure enzymes and other non-antibody proteins, use the Protein Stabilizing Cocktail for storage and preservation. Thermo Scientific Antibody Stabilizer and Storage Solutions. Page Ethylene Glycol (50% solution), 200 ml Pierce Peroxidase Conjugate Stabilizer, 25 ml Guardian Peroxidase Conjugate Stabilizer/Diluent, 200 ml Guardian Peroxidase Conjugate Stabilizer/Diluent, 1 L Protein Stabilizing Cocktail (4X), 10 ml To order or for technical support, contact your local office or distributor. See pages -2, 3.

7 Secondary Antibodies Clean-Blot IP Detection Reagents and Kit No more interference from the IP antibody. Antibody bands often mask target proteins when performing Western blots on immunoprecipitated samples. Thermo Scientific Clean-Blot IP Detection Reagents are unique HRP and AP conjugates that reveal your target protein, allowing clear, specific Western blot detection from immunoprecipitation (IP) experiments and tissue extracts without any interference from denatured IgG. Whereas conventional secondary antibodies recognize both denatured and native IgG, the Clean-Blot* IP Reagents bind to only native IgG. So unmask your results by simply substituting the secondary antibody with Clean-Blot IP Detection Reagents for clear Western blots. Versatile recognizes most native antibodies independent of the host species (Table) Compatible clear results with IPs performed using Protein A, Protein G or anti-igg agarose beads and any blocking buffer (e.g., milk, BSA or Thermo Scientific SuperBlock or StartingBlock Blocking Buffers) Cost effective eliminates the need to immobilize IgG and purchase separate kits specific for the primary antibody species; membranes can be stripped and reprobed when chemiluminescent substrate is used Flexible use any HRP or AP substrate, including chemiluminescent, fluorescent or colorimetric substrates Easy to use simply replace the conventional secondary antibody with the Clean-Blot IP Detection Reagents in your Western blotting protocol Unobstructed detection clear IP/Western blot results without interference from denatured IgG bands These IP Detection Reagents are the perfect substitute for traditional secondary antibody conjugates. The unique conjugates recognize most primary antibodies, independent of the host species, and can be used with IPs performed using Protein A or G agarose resins. This versatility eliminates the need to buy separate detection kits based on primary antibody species. Our conjugates are conveniently stored at 2-8 C and are compatible with any HRP or AP substrate, including Thermo Scientific Pierce ECL, SuperSignal Chemiluminescent and Lumi-Phos* WB Substrates. For added convenience, the HRP conjugate is available in a kit that contains StartingBlock* T20 Blocking Buffer and Pierce ECL Chemiluminescent Substrate. Thermo Scientific Clean-Blot IP Detection Reagents recognize the various polyclonal antibodies and the specific monoclonal antibodies listed. To determine specific antibody compatibility, perform a dot-blot analysis. Species Bovine IgG 2 Goat IgG 2 Monoclonal Isotype(s) Human IgG 1, IgG 2, IgG 4 Mouse IgG 2a, IgG 2b, IgG 3 Rat IgG 2c Sheep IgG 2 Lane 1 Lane 2 Lane 3 Lane 4 Lane 5 Lane Wash Elute Immunoprecipitation (IP) and Western blot experiments demonstrate specificity of the Thermo Scientific Clean-Blot IP Detection Reagent (HRP). Lane 1. A431 total cell extract expressing p53 (positive control), Lanes 2 and 3. No-lysate negative control of IP wash (Lane 2) and elution (Lane 3) fractions, Lanes 4-. Complete IP experiment of wash (Lane 4) and elution (Lanes 5 and ) fractions. Lanes 1-5 were probed with Clean-Blot IP Detection Reagent (HRP) and Lane was detected with goat antimouse IgG HRP-conjugate (GAM-HRP). Wash Elute Elute + Control - Control Immunoprecipitation Clean-Blot IP Detection Reagent (HRP) 2.5 ml Sufficient reagent for approximately 100 Western blots Clean-Blot IP Detection Kit (HRP) Sufficient reagents for approximately 2,000 cm 2 of membrane. Includes: Clean-Blot Detection Reagent (HRP) StartingBlock T20 (TBS) Blocking Buffer Pierce ECL Detection Reagent 1, Peroxide Solution Pierce ECL Detection Reagent 2 Kit 2.5 ml 1 L 125 ml 125 ml Luminol Enhancer Solution Clean-Blot IP Detection Reagent (AP) Sufficient reagent for approximately 100 Western blots. 2.5 ml Patent, see Patent Index. To view data on our Clean-Blot Detection Reagents, visit p53 Additional dry ice and/or freight charges Additional hazardous handling charge *Trademark, see Trademark Index To order or for technical support, contact your local office or distributor. See pages -2,

8 Secondary Antibodies Fluorescent Fluorophore-Labeled Probes For many years, fluor-labeled secondary antibodies and other probes have been used in a small number of cell biology applications such as flow cytometry (FC), fluorescence-activated cell sorting (FACS) and immunohistochemistry (IHC) using fluorescence microscopy. Until recently, the two most common fluorophores for labeling probes were fluorescein (fluorescein isothiocyanate, FITC) and rhodamine (tetramethyl rhodamine isothiocyanate, TRITC). In recent years, the growing demand for multiplex assays and high throughput have driven the development of many new fluorescent dyes that are brighter and more photostable and that comprise a broader range of nonoverlapping spectra. Together with the invention of more sensitive instrumentation to detect and analyze fluorescence data, these new fluors provide for extremely powerful analyses in all types of protein detection techniques. We have met the demand for high-performance dyes by developing Thermo Scientific DyLight Fluors, which provide superior performance to fluorescein and other traditional fluorophores. Available as activated dyes for labeling (see Catalog Section 9) and as conjugated secondary antibodies and biotin-binding proteins (see below), DyLight* Fluors facilitate multiplexing analysis in several nonoverlapping excitation/emission spectra. DyLight Fluor Antibody and Streptavidin Conjugates Bright alternatives to Alexa*, CyDye* and LI-COR Fluorescent Dye conjugates. Thermo Scientific DyLight Fluorescent Secondary Antibodies, Streptavidin and NeutrAvidin Protein are high-performance conjugates for use as secondary molecular probes in fluorescence microscopy, flow cytometry, Western blotting, ELISA, high-content screening and other array platforms. The goat anti-mouse IgG and anti-rabbit IgG conjugates provide for detection of these popular species of primary antibodies. The Streptavidin and NeutrAvidin Protein conjugates provide for detection of biotinylated targets. All four proteins are offered as highly functional conjugates with each of six different DyLight Fluorescent Dyes having absorption spectra ranging from 493 nm to 777 nm and bright emission spectra that match the principal output wavelengths of common fluorescence instrumentation. DyLight Conjugates exhibit higher fluorescence intensity and photostability than Alexa, CyDye and LI-COR dyes in many applications and remain highly fluorescent over a broad ph range (ph 4-9). Spectral properties of Thermo Scientific DyLight Fluorescent Dyes for which conjugated secondary antibodies are available. DyLight Dye Emission Ex/Em ε Spectrally Similar Dyes 488 Green 493/518 70,000 Alexa 488, fluorescein and FITC 549 Yellow 52/57 150,000 Alexa 54, Alexa 555, Cy3 and TRITC 594 Red 593/18 80,000 Alexa Fluor 594 and Texas Red* 49 Red 54/73 250,000 Alexa 47 and Cy5 80 Near IR 92/ ,000 Alexa 80 and Cy Near IR 777/ ,000 IRDye 800 Excitation and emission maxima in nanometers Molar extinction coefficient (M -1 cm -1 ) Fluorescence imaging with Thermo Scientific DyLight 488 Secondary Antibody. Adipose-derived mesenchymal stem cells differentiated for 24 hours and detected with the Thermo Scientific Cellomics Neurite Outgrowth Kit (K , see page 415). Thermo Scientific DyLight Fluor Wavelength (nm) Emission spectra for Thermo Scientific DyLight Fluors for which conjugated secondary antibodies are available. General DyLight Fluorophore Bright fluorescence high-intensity emission provides outstanding sensitivity and requires less conjugate Narrow emission spectra negligible bleed-through between fluorophore channels enables multi-color detection Excellent photostability exceptional resistance to photobleaching enables fluorescence imaging under the most demanding conditions (e.g., structured illumination, 4Pi microscopy) Buffer stability conjugated fluorophores are completely stable at ph 4-9 Instrument compatibility excitation and emission spectra correspond with filter sets and laser settings of all popular fluorescence instrumentation DyLight Conjugate Anti-mouse and anti-rabbit antibodies polyclonal goat antibodies provide species-specific detection of the entire target IgG class (heavy and light chains, H+L) Streptavidin and NeutrAvidin Protein choose the biotin-binding protein that is best for your application Optimized for conjugate performance reagents, labeling (dye:protein ratio) and purification methods are optimized to provide the highest possible binding function and overall fluorescence intensity Consistent and stable all conjugates are stable for 1 year at 4 C, providing consistent performance for the duration of use 134 To order or for technical support, contact your local office or distributor. See pages -2, 3.

9 Secondary Antibodies Fluorescent A. Thermo Scientific DyLight Fluor B. Thermo Scientific DyLight Fluor Wavelength (nm) Excitation and emission spectra for Thermo Scientific DyLight Fluors for which conjugated secondary antibodies and biotin-binding proteins are available (see below). Panel A. Green, orange and red visible fluors. Panel B. Near-infrared and infrared fluors. Fluorophore-Conjugated Secondary Antibodies Traditional FITC (fluorescein) and other conjugates for cell sorting and other methods. Choose from our wide selection of secondary antibodies that are labeled with fluorescein (FITC), rhodamine (TRITC), Texas Red* (a form of rhodamine), R-phycoerythrin or allophycocyanin fluorescent dyes. Find an antibody with the specificity needed for nearly any immunofluorescence experiment Wavelength (nm) Product # (Package size for these items is 1 mg at 1 mg/ml.) Description DyLight 488 DyLight 549 DyLight 594 DyLight 49 DyLight 80 DyLight 800 Goat Anti-Mouse IgG (H+L) Goat Anti-Rabbit IgG (H+L) Streptavidin NeutrAvidin Protein Characteristics of traditional fluors. Fluorophore Emission Color Ex/Em (nm) ε Fluorescein Green 491/518 8,000 Rhodamine Yellow 541/572 5,000 R-Phycoerythrin Yellow 480, 545, 55/578 ~ 2 x 10 Texas Red Red 59/15 80,000 Allophycocyanin Red 20, 45/0 ~ 7 x 10 Molar extinction coefficient (M -1 cm -1 ) Product # and Package Size Description Specificity Source Fluorescein Rhodamine Texas Red R-Phycoerythrin Allophycocyanin Anti-CHICKEN Chicken IgY (H+L) Rabbit mg Anti-GOAT Goat IgG (H+L) Mouse (min x HnMsRb Sr Prot) 1 mg 1 mg 1 mg Goat IgG (H+L) Rabbit mg 1.5 mg 1.5 mg Goat IgG [F(ab ) 2 ] Rabbit mg Goat IgG (Fc) Rabbit mg Goat IgG (H+L) Donkey (min x ChGuHaHnHs F(ab ) 2 1 ml 0.5 ml MsRbRt Sr Prot) Anti-HAMSTER Hamster IgG (H+L) Rabbit mg See Table on page 130 for the Key to Abbreviations. Additional dry ice and/or freight charges Additional hazardous handling charge *Trademark, see Trademark Index To order or for technical support, contact your local office or distributor. See pages -2,

10 Secondary Antibodies Fluorescent Product # and Package Size Description Specificity Source Fluorescein Rhodamine Texas Red R-Phycoerythrin Allophycocyanin Anti-HUMAN Human IgG (H+L) Goat mg 2 mg 2 mg Human IgG (H+L) Goat (min x BvHsMs Sr Prot) 1.5 mg 1.5 mg 1.5 mg Human IgM (Fc5µ) Goat mg Human IgA (α) Goat mg Human IgG (Fc) Rabbit mg Human IgA + IgG + IgM (H+L) Goat F(ab ) 2 1 mg Anti-MOUSE Mouse IgG (H+L) Goat mg 2 mg 2 mg Mouse IgG (H+L) Goat (min x BvHnHs Sr Prot) 1.5 mg 1.5 mg 1.5 mg Mouse IgG [F(ab ) 2 ] Goat mg Mouse IgG (Fc) Goat mg 2 mg Mouse IgM (µ) Goat mg Mouse IgG (Fcγ) Goat (subclasses 1+2a+2b+3) 1 mg 1 ml 0.5 ml (min x BvHnRb Sr Prot) Mouse IgG (Fcγ) Goat subclass 1 specific 0.5 ml 0.3 ml (min x BvHnRb Sr Prot) Mouse IgG (Fcγ) Goat subclass 2a specific 0.5 mg 0.5 ml 0.3 ml (min x BvHnRb Sr Prot) Mouse IgG (H+L) Rabbit mg 1.5 mg 1.5 mg Mouse IgG [F(ab ) 2 ] Rabbit mg Mouse IgG (Fc) Rabbit mg Mouse IgM (µ) Rabbit mg Mouse IgG (H+L) Goat 3155 (min x BvHnHs Sr Prot) F(ab ) 2 1 mg Anti-RABBIT Rabbit IgG (H+L) Donkey (min x BvChGtGuHaHn 0.5 mg 0.5 mg 0.5 mg HsMsRtSh Sr Prot) Rabbit IgG (H+L) Goat mg 2 mg 2 mg Rabbit IgG (H+L) Goat (min x Hn Sr Prot) 1.5 mg 1.5 mg 1.5 mg Rabbit IgG [F(ab ) 2 ] Goat mg Rabbit IgG (H+L) Mouse (min x GtHnMsSh Sr Prot) 1 mg Rabbit IgG (H+L) Goat F(ab ) 2 1 mg Rabbit IgG (H+L) Goat (min x HnMsRt Sr Prot) F(ab ) 2 1 mg 1 ml 0.5 ml Anti-RAT Rat IgG (H+L) Goat mg 2 mg Rat IgG (Fc) Goat mg Anti-SHEEP Sheep IgG (H+L) Rabbit mg See Table on page 130 for the Key to Abbreviations. 13 To order or for technical support, contact your local office or distributor. See pages -2, 3.

11 Antibodies Anti-Label and Anti-Tag Miscellaneous Fluorophore Conjugates Product # and Package Size Description Fluorescein Rhodamine Texas Red R-Phycoerythrin Allophycocyanin Anti-Fluorescein (FITC) mg 0.5 mg Mouse IgG, Whole Molecule mg Avidin mg 1 mg Biotin mg NeutrAvidin Protein mg Streptavidin mg 1 mg 1 mg 1 ml 0.5 ml Anti-Label and Anti-Tag Antibodies Product # and Package Size Specificity Description Unconjugated Biotin Fluorophore Anti-Biotin Goat Polyclonal Anti-Biotin mg Anti-Avidin Goat Polyclonal Anti-Avidin mg Anti-Fluorescein (FITC) Mouse Monoclonal (IgG 1 ) Anti-Fluorescein mg Anti-GST Mouse Monoclonal (IgG 1, Clone G ) Anti-Glutathione S-Transferase Monoclonal Primary Antibodies mg 3187 Rhodamine, 0.5 mg Texas Red*, 0.5 mg Product # Specificity Host Clone Isotype Format Actin Reacts with rabbit alpha actin, human actin and most other actins found in eukaryotic cells including beta, gamma and delta. No reactivity is seen with other components of the cytoskeleton. Applications: Enzyme immunohistochemistry in frozen sections and immunoblotting. Mouse ZSA1 IgM Ascites 1 ml Human Serum Albumin (HSA) Mouse ZMHSA1 IgG 1 Purified 0.5 mg Specific for human serum albumin. It is suitable for ELISA techniques or radiolabeling. Antibody Handbook ANTIBODIES Antikörper Anticuerpos Anticorpi Anticorps Anticorpos Thermo Scientific Pierce Antibody Handbook The antibodies presented in this handbook are just a small portion of our total offering. Visit to select from more than 30,000 antibodies and antibodyrelated literature. Additional dry ice and/or freight charges Additional hazardous handling charge *Trademark, see Trademark Index To order or for technical support, contact your local office or distributor. See pages -2,

12 Detection Probes His Tag and Phosphoprotein Specific HisProbe-HRP Conjugate and Detection Kit HRP Western blotting probe takes advantage of the affinity of histidine for the Ni 2+ cation. Thermo Scientific HisProbe-HRP is a nickel (Ni 2+ )-activated derivative of horseradish peroxidase (HRP). This product has been optimized for direct detection of recombinant histidine-tagged proteins and other histidine-rich proteins. The active ligand is a tridentate chelator that allows Ni 2+ to be bound in active form for subsequent interaction and detection of target molecules. The active chelator has similar binding capabilities to that reported for iminodiacetic acid, which has long been used for immobilized metal affinity chromatography (IMAC). Ni 2+ HisHisHisHisHisHis His Protein Substrate HRP Ni 2+ Signal Detection of histidine-tagged fusion proteins with Thermo Scientific HisProbe-HRP. PhosphoProbe-HRP Reagents and Detection Kit Novel chemistry enables specific detection of phosphorylated protein. Thermo Scientific PhosphoProbe-HRP is an iron (Fe 3+ )-activated derivative of horseradish peroxidase (HRP) for microplate or membrane detection of phosphorylated proteins. PhosphoProbe-HRP exhibits two distinct binding specificities, one of which is phosphate (R-PO 3 )- specific. The other binding specificity is related to a carboxyl-containing binding motif that is common to most proteins and some peptides. This carboxyl motif binding specificity can be used in a total protein detection application. A novel treatment, termed reactive chemical blocking (RCB), may be used to eliminate this carboxyl-binding motif, thus imparting exclusive specificity toward phosphate groups. PhosphoProbe-HRP, in conjunction with RCB, is a universal phosphate detection probe. PhosphoProbe-HRP has been optimized for direct detection of phosphoester molecules such as nucleotides or protein/ peptides containing phosphoserine, phosphothreonine and phosphotyrosine. Ni 2+ For detection of histidine-tagged proteins Yields lower background than anti-histidine antibodies Thermo Scientific Pierce HRP is a high-activity enzyme Stripping and reprobing is possible Compatible with Thermo Scientific SuperSignal Chemiluminescent Substrates 1515 HisProbe-HRP 2 mg 1518 SuperSignal West Pico HisProbe Kit Kit Includes: HisProbe-HRP 2 mg SuperSignal West Pico 500 ml Chemiluminescent Substrate Blocker BSA in TBS (10X) 1 x 125 ml BupH Tris Buffered Saline Packs 10 packs Surfact-Amps 20 (10%) Detergent x 10 ml ampules Patent Pending Patent, see Patent Index. 151 PhosphoProbe-HRP 2 mg 1517 PhosphoProbe Phosphoprotein Kit Detection Kit Includes: PhosphoProbe-HRP 2 mg EDC 5 g Ethylenediamine 10 g Tween*-20 1 vial Patent, see Patent Index. Related Products Page # Ethylenediamine Dihydrochloride EDC 330 PO 3 COOH Carboxyls are selectively converted to amines COOH PO 3 EDC and EDA Reactive chemical blocking step Protocol scheme for specific detecton of phosphorylated proteins with Thermo Scientific PhosphoProbe-HRP. PO 3 O NH 2 C=C N C H O C N C=C NH 2 H PO 3 HRP Fe3+ Addition of PhosphoProbe-HRP PhosphoProbe-HRP binds only to free phosphates HRP H 2 N C=C N C Fe3+ PO 3 O Phosphoprotein Phosphoprotein Phosphoprotein O C N C=C NH 2 PO 3 Fe3+ HRP 138 To order or for technical support, contact your local office or distributor. See pages -2, 3.

13 Antibodies Nonspecific Normal Sera and Nonspecific Antibodies Normal serum is the term used to describe serum from an animal that has not been immunized to produce antibodies against specific antigens. Thus, normal serum is the opposite of anti-serum. Normal sera contain the usual complement of serum proteins, including the various classes of immunoglobulins that exist in healthy animals of the particular species. Normal serum is frequently used for blocking or saturating generalized binding interactions for immunodetection methods, especially those involving tissue samples such as immunohistochemistry. Normal sera are also useful as controls for testing general and specific antibody purification methods. Nonspecific antibodies are immunoglobulins or immunoglobulin fractions purified from normal sera. Presumably they do not contain antibodies specific for particular antigens that are targets of primary antibodies in any experimental system. Such nonspecific immunoglobulins (and other purified serological proteins) are useful as assay standards, experimental controls and blocking agents in a variety of immunodetection methods. Fluorescein-, biotin- and enzymeconjugated forms provide alternative options for using these proteins as controls and standards. Immunoglobulins and Serological Proteins Human IgA, Serum 2 mg Human IgA, Serum-Fluorescein Conjugated 1 mg Human IgG, Whole Molecule 10 mg 3114 Human IgM (myeloma), Whole Molecule 2 mg 3145 Human IgG, Whole Molecule- 1 mg Peroxidase Conjugated Mouse IgG, Whole Molecule 5 mg Mouse IgG, Whole Molecule- Fluorescein Conjugated 1 mg Gamma Globulins Mouse Gamma Globulin 10 mg Rabbit Gamma Globulin 10 mg Rat Gamma Globulin 10 mg Mouse IgG, F(ab ) 2 Fragment 2 mg Mouse IgG, Fc Fragment 1 mg Mouse Transferrin-Peroxidase Conjugated 1 mg Rabbit IgG, Whole Molecule 10 mg 3182 Rabbit IgG Immunoglobulin-Biotinylated 5 mg Rat IgG, Whole Molecule 10 mg Sheep IgG, Whole Molecule 10 mg Goat Gamma Globulin 10 mg Human Gamma Globulin 10 mg Normal Sera Normal Goat Serum 2 ml Normal Goat Serum 10 ml Normal Horse Serum 2 ml 3187 Normal Human Serum 2 ml Normal Mouse Serum 2 ml Normal Mouse Serum 5 ml Normal Rabbit Serum 2 ml Normal Rabbit Serum 5 ml Normal Rat Serum 2 ml Normal Swine Serum 2 ml Additional dry ice and/or freight charges Additional hazardous handling charge *Trademark, see Trademark Index To order or for technical support, contact your local office or distributor. See pages -2,

14 Proteins Antibody-Binding Native Protein A Binds specifically to the Fc region of immunoglobulin molecules, especially IgG. Native Protein A purified from Staphylococcus aureus 4.7 kda (unconjugated); 42 kda by SDS-PAGE Contains four Fc-binding domains Best for polyclonal IgG from rabbit, pig, dog and cat serum Poor for Mouse IgG 1, human IgG 3, rat, goat and cow Protein A 5 mg Biotinylated Protein A 1 mg Recombinant Protein A No enterotoxins present, as there may be from Staphylococcusderived Protein A. Recombinant Protein A expressed in E. coli 45 kda (unconjugated) Contains four Fc-binding domains Recombinant Protein G Useful for a variety of immunological and biochemical techniques. Recombinant Protein expressed in E. coli 21. kda (unconjugated); kda by SDS-PAGE Contains two Fc-binding domains Best for IgG from mouse, human, cow, goat and sheep Poor for guinea pig, pig, dog and cat Does not bind human IgM, IgD, IgA Recombinant Protein A/G Produced by gene fusion of the Fc binding domains of Protein A and Protein G. Recombinant protein expressed in E. coli 50.5 kda (unconjugated); kda by SDS-PAGE Contains four Fc-binding domains from Protein A and two from Protein G Protein A 5 mg Protein A, Peroxidase Conjugated 1 mg Protein G 5 mg Biotinylated Protein G 0.5 mg Protein G, Peroxidase Conjugated 0.5 mg 2118 Protein A/G 5 mg Protein A/G, Alkaline Phosphatase Conjugated 0.5 mg Protein A/G, Peroxidase Conjugated 0.5 mg Recombinant Protein L Binds a wider range of Ig classes and subclasses, including all classes of IgG and single-chain variable (ScFv) and Fab fragments. Recombinant protein expressed in E. coli 35.8 kda (unconjugated) Contains four IgG-binding domains Binds to immunoglobulins that contain kappa light chains (kappa I, III and IV from human and kappa I from mouse) Protein L 1 mg Protein L, Peroxidase Conjugated 0.5 mg Biotinylated Protein L 0.5 mg 140 To order or for technical support, contact your local office or distributor. See pages -2, 3.

15 Proteins Biotin Detection Biotin-Binding Proteins The highly specific interaction between biotin (a vitamin) and avidin (hen egg white protein) or streptavidin (a bacterial protein) has been exploited for use in many protein and nucleic acid detection methods. The most common application for Western blotting and other detection methods is to label primary or secondary antibodies with biotin for subsequent detection with avidin protein that has been conjugated with horseradish peroxidase. Because the biotin label is stable and small (244 daltons), it rarely interferes with the function of the labeled antibody or protein and provides for development of robust and highly sensitive assays. Streptavidin, a bacterial protein, has nearly the same biotin-binding properties as avidin (K a = M -1 ), but causes less nonspecific lectin binding because it is not glycosylated like avidin. However, streptavidin Avidin Products Convenient conjugates for assay detection. Avidin is a tetrameric glycoprotein (MW 7,000) purified from chicken egg white. The highly specific interaction of avidin with biotin makes it a useful tool in designing nonradioactive detection systems. The extraordinary affinity of avidin for biotin (K a = M -1 ) allows biotin-labeled molecules to be detected with excellent sensitivity and specificity. contains a RYD motif, which is a bacterial recognition sequence that can cause background binding with certain samples. Thermo Scientific NeutrAvidin Protein is an exclusive, deglycosylated form of avidin that avoids the drawbacks of both native avidin and streptavidin. Comparison of biotin-binding proteins. Avidin Streptavidin NeutrAvidin Protein Molecular Weight 7K 53K 0K Biotin-binding Sites Isoelectric Point (pl) Specificity Low High Highest Affinity for Biotin (Kd) M M M Nonspecific Binding High Low Lowest In addition to the Thermo Scientific Avidin, Streptavidin and NeutrAvidin Proteins and Conjugates described here, we offer a wide selection of biotin-labeled antibodies (see p. 130 and Catalog Section 12) and a complete selection of biotinylation kits and reagents (Catalog Section 9). Avidin is more soluble than streptavidin and has an isoelectric point (pi) of It is also more economical than streptavidin, and is commonly used in signal amplification systems such as the ABC system (see Immunohistochemistry products, page 188). Bruch, R.C. and White, III, H.B. (1982). Biochemistry 21, Chaiet, I. and Wolf, F.J. (194). Arch. Biochem. Biophys. 10, 1-5. Gitlin, G., et al. (1987). Biochem. J. 242, Savage, M.D., et al. (1992). Avidin-Biotin Chemistry: A Handbook. Rockford, Illinois: Pierce Chemical Company. Wilchek, M. and Bayer, E.A. (1983). Anal. Biochem. 171, Zuk, P.A. and Elferink, L.A. (2000). J. Biol. Chem. 275, Features Applications Avidin Avidin 10 mg 20 mg Hen egg white glycoprotein, affinity-purified, salt-free, lyophilized powder µg biotin bound/mg avidin Isoelectric point of Stable over a wide range of ph and temperatures Horseradish Peroxidase Conjugated 2 mg Purified using special affinity techniques to Horseradish Peroxidase Conjugated 5 mg eliminate nucleic acids 1-2 moles HRP/mole avidin 5-10 µg biotin bound/mg protein 80 peroxidase units/mg protein Alkaline Phosphatase Conjugated 100 units Homogeneous by SDS-PAGE Purified using special affinity techniques to eliminate nucleic acids ~1 mole alkaline phosphatase/mole avidin One unit = 1.0 µmol of p-nitrophenol liberated from p-nitrophenylphosphate per minute at 37 C, ph Fluorescein (FITC) Conjugated 5 mg Fluorophore-labeled avidin Ex/Em: 490 nm/520 nm No free fluorescein ~3.5 moles fluorescein/mole avidin R-Phycoerythrin Conjugated 1 mg Fluorophore-labeled avidin Ex/Em: nm/574 nm Immunoassay reagent when bound to biotinylated enzymes or when conjugated to enzymes Blocking protein for biotin-rich tissue sections (use at 0.1% for inhibition of endogenous biotin) Use in immunohistochemistry where endogenous phosphatase is a problem Western blotting Use for immunohistochemistry where high levels of endogenous peroxidase is a problem Western blotting ELISA Fluorescence-activated cell sorting (FACS) Histochemical staining Fluorescence-activated cell sorting (FACS) Histochemical staining Additional dry ice and/or freight charges Additional hazardous handling charge *Trademark, see Trademark Index To order or for technical support, contact your local office or distributor. See pages -2,

16 Proteins Biotin Detection NeutrAvidin Products For ultralow nonspecific binding compared to avidin or streptavidin! Achieve better assay results with the low nonspecific binding properties of Thermo Scientific NeutrAvidin Protein. NeutrAvidin* Biotin-Binding Protein is a deglycosylated form of avidin, so lectin binding is reduced to undetectable levels without losing biotin-binding affinity (K a = M -1 ). 1 NeutrAvidin Biotin-Binding Protein offers the advantage of a neutral pi to minimize nonspecific adsorption, along with lysine residues that remain available for derivatization or conjugation through amine-reactive chemistries. The molecular weight of NeutrAvidin Biotin-Binding Protein is approximately 0,000. The specific activity for biotin-binding is approximately 14 µg/mg of protein, which is near the theoretical maximum activity. Near-neutral pi (.3) and no glycosylation, unlike avidin No RYD recognition sequence, unlike streptavidin Generally lower nonspecific binding than avidin and streptavidin Much lower price than streptavidin 1. Hiller, Y., et al. (1987). Biochem. J. 248, Claypool, S.M., et al. (2002). J. Biol. Chem. 27, Glover, B.P. and McHenry, C.S. (2001). Cell 105, Guo, Y., et al. (2001). J. Biol. Chem. 27, Unson, M.D., et al. (1999). J. Clin. Microbiol. 37, Wojciechowski, M., et al. (1999). Clin. Chem. 45, Features NeutrAvidin Protein 10 mg pi that has been reduced to a neutral state Deglycosylated, so lectin binding is reduced to undetectable levels Can be used as a biotin blocking agent in tissues for histochemistry µg biotin bound/mg NeutrAvidin Protein Horseradish Peroxidase Conjugated 2 mg Better signal:noise ratio in assay systems 1-2 moles HRP/mole NeutrAvidin Protein 3-8 µg biotin bound/mg conjugate High Sensitivity HRP Conjugate 0.5 ml Stabilized solutions of poly-hrp NeutrAvidin Conjugates Supplied as 1 mg/ml Excellent for ELISA, Western blotting and histochemical staining procedures requiring highest possible sensitivity High Sensitivity HRP Conjugate 5 ml Stabilized solutions of poly-hrp NeutrAvidin Conjugates Supplied as 1 mg/ml Excellent for ELISA, Western blotting and histochemical staining procedures requiring highest possible sensitivity Alkaline Phosphatase Conjugated 2 mg Lower nonspecific binding than streptavidin conjugates Better signal:noise ratio in assay systems 3-8 µg biotin bound/mg conjugate 3100 Fluorescein Conjugated 5 mg Fluorophore-labeled NeutrAvidin Protein Excitation: 490 nm; Emission 520 nm 2 moles fluorescein/mole NeutrAvidin Protein DyLight 488 Conjugated 1 mg in 1 ml Fluorophore-labeled NeutrAvidin Protein Excitation: 493 nm; Emission 518 nm Tested for signal-to-background ratio DyLight 549 Conjugated 1 mg in 1 ml Fluorophore-labeled NeutrAvidin Protein Excitation: 550 nm; Emission 58 nm Tested for signal-to-background ratio DyLight 49 Conjugated 1 mg in 1 ml Fluorophore-labeled NeutrAvidin Protein Excitation: 4 nm; Emission 74 nm Tested for signal-to-background ratio DyLight 80 Conjugated 1 mg in 1 ml Fluorophore-labeled NeutrAvidin Protein Excitation: 82 nm; Emission 715 nm Tested for signal-to-background ratio DyLight 800 Conjugated 1 mg in 1 ml Fluorophore-labeled NeutrAvidin Protein Excitation: 770 nm; Emission 794 nm Tested for signal-to-background ratio Maleimide Activated 5 mg Prepare NeutrAvidin conjugates of proteins/peptides Reacts spontaneously with free sulfhydryls in the ph range of moles maleimide/mole NeutrAvidin Protein Looking for related Avidin, Streptavidin or NeutrAvdin Protein products? For Location in Catalog Coated microplates This section, page 172 Affinity resins (e.g., agarose) Catalog Section 2 Biotin-labeling reagents Catalog Section 9 Sandwich ELISA Kits Catalog Section To order or for technical support, contact your local office or distributor. See pages -2, 3.

17 Proteins Biotin Detection Streptavidin Products Wide selection of conjugates for almost any biotin-based assay. Originally isolated from Streptomyces avidinii, streptavidin is a tetrameric biotin-binding protein. Recombinant streptavidin is smaller than the native protein (MW 53,000) and has a more neutral isoelectric point (pi.8-7.5). Streptavidin is carbohydrate-free and much less soluble in water than avidin, resulting in high binding affinity, capacity, and specificity for biotinylated molecules. Thermo Scientific Streptavidin Conjugates are useful for secondary detection in Western blotting, ELISA and cell and tissue staining. Features Applications Streptavidin 1 mg Lyophilized, stable powder Immunoassay reagent when bound to biotinylated Streptavidin 5 mg No carbohydrate enzymes or when conjugated to enzymes Much less soluble in water than avidin Blocking protein for biotin-rich tissue sections µg biotin bound/mg of protein (use at 0.1% for inhibition of endogenous biotin) Recombinant Can be used with biotinylated enzymes (Product # or 29139) 2112 Horseradish Peroxidase Conjugated 1 mg 1-2 moles HRP/mole streptavidin Horseradish Peroxidase Conjugated 2 mg 100 peroxidase units/mg conjugate Lyophilized, stable powder Horseradish Peroxidase Conjugated 5 mg -9 µg biotin bound/mg conjugate High Sensitivity HRP Conjugate 0.5 ml Stabilized solutions of poly-hrp High Sensitivity HRP Conjugate 5 ml streptavidin conjugates Product # and are High Sensitivity HRP Conjugate, 2 ml standard format (1 mg/ml) Pre-diluted (10 µg/ml) Product # is pre-diluted (10 µg/ml format) Alkaline Phosphatase Conjugated 1 mg 3 µg biotin bound/mg conjugate Alkaline Phosphatase Conjugated 3 mg 100 phosphatase units/mg conjugate Fluorescein (FITC) Conjugated 1 mg Fluorophore-labeled streptavidin Ex/Em: 490 nm/520 nm 3-5 moles FITC/mole streptavidin Rhodamine (TRITC) Conjugated 1 mg Fluorophore-labeled streptavidin Excitation: nm/ nm Emission: 575 nm 1-3 moles TRITC/mole streptavidin 2124 Texas Red* Conjugated 1 mg Fluorophore-labeled streptavidin Ex/Em: 595 nm/15 nm 2127 R-Phycoerythrin Conjugated 1 ml Fluorophore-labeled streptavidin Ex/Em: nm/574 nm 2129 Allophycocyanin Conjugated 0.5 ml Fluorophore-labeled streptavidin Ex/Em: 50 nm/0 nm DyLight 488 Conjugated 1 mg in 1 ml Fluorophore-labeled streptavidin Ex/Em: 493 nm/518 nm Tested for signal-to-background ratio DyLight 549 Conjugated 1 mg in 1 ml Fluorophore-labeled streptavidin Ex/Em: 550 nm/58 nm Tested for signal-to-background ratio DyLight 49 Conjugated 1 mg in 1 ml Fluorophore-labeled streptavidin Ex/Em: 4 nm/74 nm Tested for signal-to-background ratio DyLight 80 Conjugated 1 mg in 1 ml Fluorophore-labeled streptavidin Ex/Em: 82 nm/715 nm Tested for signal-to-background ratio DyLight 800 Conjugated 1 mg in 1 ml Fluorophore-labeled streptavidin Ex/Em: 770 nm/794 nm Tested for signal-to-background ratio Hydrazide Activated 2 mg Attaches streptavidin to oxidized carbohydrate residues on glycoproteins 4 moles hydrazide/mole streptavidin Maleimide Activated 1 mg Reacts spontaneously with free sulfhydryls in the ph range of moles maleimide/mole streptavidin Histochemical staining Western blotting Conti, L.R., et al. (2001). J. Biol. Chem. 27, ELISA and Western blotting procedures requiring highest possible sensitivity Histochemical staining requiring high sensitivity and low background Histochemical staining Western blotting Harriman, G.R., et al. (1999). J. Immunol. 12, Nielsen, P.K., et al. (2000). J. Biol. Chem. 275, Histochemical staining Fluorescence-activated cell sorting (FACS) Histochemical staining Fluorescence-activated cell sorting (FACS) Histochemical staining; can be used in double staining methods Fluorescence-activated cell sorting (FACS) Histochemical staining Fluorescence-activated cell sorting (FACS) Histochemical staining Fluorescence-activated cell sorting (FACS) Histochemical staining Fluorescence-activated cell sorting (FACS) Western blotting ELISA Histochemical staining Fluorescence-activated cell sorting (FACS) Western blotting ELISA Histochemical staining Fluorescence-activated cell sorting (FACS) Western blotting ELISA Histochemical staining Fluorescence-activated cell sorting (FACS) Western blotting ELISA Histochemical staining Fluorescence-activated cell sorting (FACS) Western blotting ELISA Used to create immunoassay reagents Localize glycoproteins on blot transfers, followed by detection with a biotinylated enzyme Create protein-interaction reagents Prepare streptavidin conjugates of proteins or peptides Additional dry ice and/or freight charges Additional hazardous handling charge *Trademark, see Trademark Index To order or for technical support, contact your local office or distributor. See pages -2,

18 Conjugates Biotin Biotin and Biotin Conjugates Features Biotin 1 g Free biotin is used in the elution buffer to purify biotinylated compounds from monomeric avidin columns Can be used as a reference standard for the HABA dye assay when measuring degree of biotinylation Biotin-Fluorescein 5 mg Excitation 492 nm, Emission 510 nm Color: Greenish-yellow Biotinylated Bovine Serum Albumin (BSA) Biotinylated Horseradish Peroxidase (HRP) Biotinylated Alkaline Phosphatase (AP) Looking for related biotin products? 25 mg Useful as a control in ELISA, immunoblotting and immunohistochemical studies Supplied as a lyophilized powder with 8-12 moles of biotin per mole of BSA 5 mg Offers excellent sensitivity and is recommended for use in sandwich techniques that utilize Avidin, Streptavidin or NeutrAvidin Protein Unit is defined as amount of Biotinylated-HRP required to form 1 µmol of purpurogallin from pyrogallol in 20 seconds at 20 C 1 mg One unit equals the amount of protein needed to hydrolyze 1.0 µmol of p-nitrophenyl phosphate per minute at 25 C in the following buffer: 0.1 M glycine, 1.0 mm ZnCl 2, 1.0 mm MgCl 2, mm PNPP, ph Biotinylated β-galactosidase 100 units Used with ONPG for histochemical and immunoblotting applications One unit = 1 µmol o-nitrophenyl-β-d-galactopyranoside (ONPG) hydrolyzed per minute at 37 C, ph Biotinylated Protein G 0.5 mg Useful as a substitute for biotinylated secondary antibodies Can detect or locate immunoglobulins on cell surface or in tissue Biotinylated Protein A 1 mg Useful as a substitute for biotinylated secondary antibodies Can detect or locate immunoglobulins on cell surface or in tissue Biotinylated Protein L 0.5 mg Binds to all classes of IgG (IgG, IgM, IgA, IgE and IgD) with particular kappa light chains Binds to kappa light chains without interfering with antigen binding Binds single-chain variable fragments (ScFv) 3182 Biotinylated Rabbit IgG 5 mg Detect low concentrations of Fc receptors or anti-immunoglobulin antibodies on cells or in tissue Supplied in PBS or HBS (10 mm HEPES, 0.15 M NaCl, ph 7.8) containing 0.04% sodium azide Biotinylated Goat Anti-Avidin 0.5 mg Binds to avidin either through its antigenic or biotin-binding sites Can be used to amplify a fluorescent signal Ideal for tissue staining or cell sorter analysis applications For Location in Catalog Biotin-labeled Secondary Antibodies This section, page 129 Affinity Resins (e.g., agarose) Catalog Section 2 Biotin-labeling Reagents Catalog Section 9 Sandwich ELISA Kits Catalog Section 12 Filter Paper for Blotting Clean, uniform, medium-thick (0.83 mm) paper for protein blotting methods Protects gel and transfer membranes from transfer cassette sponges 8800 Western Blotting Filter Paper 8 cm x 10.5 cm 100 sheets/pkg. 144 To order or for technical support, contact your local office or distributor. See pages -2, 3.

19 Western Blotting Membranes Thermo Scientific Products for Western Blotting Nitrocellulose Membranes Membranes with high surface area and excellent uniformity provide superior protein binding characteristics Two different pore specifications (0.2 µm and 0.45 µm) for experiments with small or large proteins Three different sheet sizes, including economically priced rolls Ideal membrane for chemiluminescent Western blotting Polyvinylidene Difluoride (PVDF) Membranes Tough membranes provide maximum binding capacity for hydrophobic proteins Compatible with most organic solvents, acids and mild bases Convenient pre-cut sheets for transfer from mini-gel or economically priced rolls Small pore-size, low-fluorescence variety provides superior performance (low background) for fluorescent detection Nylon Membranes Nitrocellulose Membrane, 0.2 µm 8 cm x 8 cm Nitrocellulose Membrane, 0.2 µm 7.9 cm x 10.5 cm Nitrocellulose Membrane, 0.2 µm 8 cm x 12 cm Nitrocellulose Membrane, 0.45 µm 8 cm x 8 cm Nitrocellulose Membrane, 0.45 µm 7.9 cm x 10.5 cm Nitrocellulose Membrane, 0.45 µm 8 cm x 12 cm Nitrocellulose Membrane, 0.45 µm 33 cm x 3 m Minimum 120 sheets when cut to 8 cm x 10 cm PVDF Transfer Membrane, 0.45 µm 10 cm x 10 cm PVDF Transfer Membrane, 0.45 µm 2.5 cm x 3.75 m Minimum 111 sheets when cut to 8 cm x 10 cm Low-Fluorescence PVDF Transfer Membrane, 0.2 µm 7 cm x 8.4 cm 15/pkg. 15/pkg. 25/pkg. 15/pkg. 15/pkg. 25/pkg. 1 roll 10/pkg. 1 roll 10/pkg. Biodyne* A Nylon Membrane: Unmodified Nylon 50% amine and 50% carboxyl groups, pi.5 Provides excellent resolution, high sensitivity and extremely low backgrounds Biodyne A Nylon Membrane 8 cm x 12 cm 0.4 µm pore size 7701 Biodyne B Nylon Membrane 8 cm x 12 cm 0.4 µm pore size 25/pkg. 25/pkg. Biodyne B Nylon Membrane: Positively charged Nylon Strong cationic quaternary ammonium groups provide positive zeta potential to ph > 10 Excellent for DNA, RNA and other negatively charged molecules Use with Thermo Scientific LightShift Chemiluminescent EMSA Kit (Product # 20148) Additional dry ice and/or freight charges Additional hazardous handling charge *Trademark, see Trademark Index To order or for technical support, contact your local office or distributor. See pages -2,

20 Western Blotting Protein Stain MemCode Reversible Protein Stain A great alternative to Ponceau S stain! For years the red Ponceau S stain has been the best option for staining before Western blotting, despite its major shortcomings. Thermo Scientific MemCode Reversible Protein Stains decrease staining time, increase staining sensitivity and enhance the immunoreactivity of antigens in subsequent Western blotting. Try these reversible protein stains for nitrocellulose and PVDF membranes and you will never use Ponceau S again. Sensitive, high-avidity, general protein stain Stain is protein-specific, avoiding interference from other biomolecules From stain to destain to band erasure in minutes Turquoise bands are easily photographed Stained bands do not fade with time Enhances Western blot detection All components are room temperature stable For additional information see page 12 Comparison of Thermo Scientific MemCode Reversible Protein Stain with Ponceau S. Ponceau S Reversible Stain Weak-binding, low sensitivity general protein stain Detection limit: 250 ng Red bands are difficult to photograph Stained protein bands fade within hours Typical staining time: 5 minutes Pierce Antibody Extender NC MemCode Reversible Protein Stain Tight-binding, higher sensitivity general protein stain Detection limit: ng Turquoise blue bands are easily photographed Turquoise bands do not fade over time, but they can be erased Typical staining time: 0 seconds Background eliminated quickly with low ph wash Reduce the amount of primary antibody used in a Western blot by three- to 100-fold. A simple 10-minute, post-transfer treatment of the target protein on nitrocellulose can reduce the amount of primary antibody used by three-, 10-, 25- and even 100-fold, while maintaining equivalent signal compared to an untreated control. Our Antibody Extender NC Promise Proper use of the Thermo Scientific Pierce Antibody Extender NC will retain post-transfer detection of your target protein on nitrocellulose membrane when using at least three times less primary antibody than you are currently using. If you do not experience a minimum of threefold reduction in primary antibody requirement with an equivalent or better performance on nitrocellulose membrane, we will refund the cost of the reagent A. MemCode Stain B. Ponceau S Stain Thermo Scientific MemCode Stain demonstrates superior sensitivity on PVDF membrane. Unstained markers were serially diluted and separated on 4-20% SDS- PAGE gels, then transferred to PVDF membrane. Blot A was stained with MemCode Stain for 1 minute and destained according to the protocol. Blot B was stained with 0.1% Ponceau S in 5% acetic acid for 5 minutes and destained according to the published protocol. Lane 10. Prestained MW Marker Mix (Product # 281) MemCode Reversible Protein Stain Kit for Nitrocellulose Membranes Sufficient material for 10 (8 cm x 8 cm) nitrocellulose membranes. Includes: Reversible Protein Stain Destain Stain Eraser MemCode Reversible Protein Stain Kit for PVDF Membranes Sufficient material to stain protein and reverse the stain from 10 (8 cm x 8 cm) PVDF membranes. Includes: Sensitizer Reversible Stain Destain Stain Eraser Kit 250 ml 1 L 250 ml Kit 250 ml 250 ml 1 L 250 ml Achieves equivalent signal while using less antibody uses threeto 100-fold less primary antibody [average primary antibody reduction factor (PAR) is 28.2-fold] Inexpensive typical savings in primary antibody more than pays for the reagent Conserves antibody, regardless of detection system works with colorimetric, chemiluminescent, HRP and AP systems Simple and ready to use fast 10-minute protocol Pierce Antibody Extender Solution NC Sufficient reagent for up to 20 nitrocellulose membranes (1,00 cm 2 ) Pierce Antibody Extender Solution NC Trial Pack Sufficient reagent to treat two nitrocellulose membranes (10 cm 2 ). 500 ml 50 ml 14 To order or for technical support, contact your local office or distributor. See pages -2, 3.

21 Western Blotting Enhancer Pierce Western Blot Signal Enhancer It s like having an intensifying screen in a bottle. Thermo Scientific Pierce Western Blot Signal Enhancer does for enzyme-/substrate-based blotting what intensifying screens do for radioactive blotting it increases the signal up to 10-fold (or one order of magnitude) in only 15 minutes. The Western Blot Signal Enhancer membrane treatment is a simple, 15-minute procedure that can be added to your current Western blotting protocol. The result is an increase in the intensity of target protein bands on the Western blot or detection of target proteins at a level that could not previously be detected. Some protein targets have resulted in a 10-fold increase in band intensity after treatment with the Western Blot Signal Enhancer compared to the typical detection protocol without treatment. Detect protein at lower levels most protein targets have shown a three- to 10-fold increase in signal intensity Enhances antibody binding to immobilized antigens signal enhancement has been demonstrated with targets such as IL-, p53, NFκB, BRCA1 and EGF Works with any substrate enhances both chemiluminescent and colorimetric detection Works with any membrane enhances signal from nitrocellulose and PVDF membrane regardless of pore size 15-minute protocol optimized to save time and improve detection capability of most proteins Ready-to-use reagents no formulating or diluting necessary Room temperature stable store reagents at your bench A. Untreated Blot B. Treated Blot Enhanced chemiluminescent detection of identical serial dilutions of IL- Panel A: before and Panel B: after treatment with Thermo Scientific Pierce Western Blot Signal Enhancer. Lane pg, Lane pg, Lane 3. 1,000 pg and Lane 4. 2,000 pg. A. Untreated Blot Enhanced chromogenic detection of identical serial dilutions of IL- Panel A: before and Panel B: after treatment with Thermo Scientific Pierce Western Blot Signal Enhancer. Lane pg, Lane pg, Lane pg, Lane pg, Lane pg, Lane. 1,000 pg and Lane 7. 5,000 pg. Ultrapure H 2 O 1. Rinse membrane after transfer with ultrapure water 4. Incubate membrane with Reagent 2 for 10 minutes on a shaker 2 Thermo Scientific Pierce Western Blot Signal Enhancer Protocol performed after transfer and before blocking. Aupperlee, M., et al. (2005). Endocrinology 14, Li, Y., et al. (2004). Blood. 104, MacMillan, D., et al. (2005). J. Cell Sci. 118, B. Treated Blot Incubate membrane with Reagent 1 for 2 minutes on a shaker Ultrapure H 2 O 5. Rinse membrane with ultrapure water (repeat 5 times) Ultrapure H 2 O 3. Rinse membrane with ultrapure water (repeat 5 times) Start your detection protocol. T otal time = 15 minute s Pierce Western Blot Signal Enhancer Kit Sufficient reagent for ten 10 cm x 10 cm blots. Includes: Enhancer Reagent ml Enhancer Reagent ml Signal enhancement of proteins on PVDF membrane has been shown to be variable from no significant enhancement for some proteins, to several-fold enhancement for others. Extend or Enhance? Choosing between Thermo Scientific Pierce Antibody Extender and Western Blot Signal Enhancer Products. Thermo Scientific Antibody Extender NC and Western Blot Signal Enhancer Products are mutually exclusive; i.e., you cannot extend your antibody and increase signal at the same time. Use Antibody Extender NC when You want a costly primary antibody to last as long as possible You have plenty of target, but the detection antibody is available in limited amount Use Western Blot Signal Enhancer when You have a low abundance of target protein (antigen), but adequate primary antibodies with which to detect it You want to obtain a stronger signal under the conditions you typically use to detect your target protein Additional dry ice and/or freight charges Additional hazardous handling charge *Trademark, see Trademark Index To order or for technical support, contact your local office or distributor. See pages -2,

22 Western Blotting Blocking Buffers Blocking Buffers for Western Blotting Before using antibodies to detect proteins that have been dotted or transferred to specific locations on a membrane, the remaining binding surface of the entire membrane must be blocked. Otherwise, the antibodies or other proteins used to probe the membrane will bind to the entire surface rather than only where the target protein is located. In principle, any protein that does not have binding affinity for the target or probe components in the Western blot procedure can be used for blocking. In practice, however, certain proteins perform better than others because they bind to the membrane more consistently or because they somehow stabilize the function of other system components. In fact, no single protein or mixture of proteins works best for all Western blot experiments, and empirical testing is necessary to obtain the best possible results for a given combination of specific antibodies, membrane type and substrate system. The accompanying figure illustrates the value of testing different blocking buffers as part of a Western blotting optimization experiment. In this example experiment, in which all other conditions were equal, different blocking buffers quenched or enhanced the sensitivity and specificity of the Western blot for individual proteins. In other cases, one blocking buffer or another might cause speckling or high background. Guide to Thermo Scientific Pierce Blocking Buffers. PBS = phosphate buffered saline; TBS = Tris buffered saline; T20 = Tween*-20 Detergent Thermo Scientific Blocking Buffers are high-quality solutions with diverse properties that provide excellent blocking performance for nearly all kinds of Western blotting experiments. As pre-formulated, quality-tested blocking buffer solutions, the products provide consistent performance, minimizing the amount of optimization needed between bottles and production lots. Cyclin B1 30-Second Exposure p53 30-Second Exposure fos 30-Second Exposure fos 5-Minute Exposure Thermo Scientific Blocking Buffer SuperBlock Buffer Blocker BLOTTO Blocker Casein Blocker BSA Importance of blocking buffer optimization. Chemiluminescent Western blot results for three proteins processed with identical conditions except for the blocking step. Each blot contains three lanes of protein corresponding to the same series of 5-fold dilutions (1:50, 1:10, 1:2). Two film exposures are shown for the fos experiment. Blocker Casein yielded the most sensitive result for Cyclin B1 protein, while SuperBlock Blocking Buffer yielded the most sensitive result for p53 and fos. In these tests involving nitrocellulose membrane, all four blockers yielded low background. Blocking Buffer Description ELISA Western Blot and Dot Blot StartingBlock Blocking Buffers Single purified protein; fast blocking; broad applicability; excellent for stripping and reprobing Western blotting applications; available in PBS and TBS with and without T20 Immunohistochemistry DNA/RNA Hybridizations SuperBlock Blocking Buffers Single purified glycoprotein; fast blocking; broad applicability; stabilizes plate-coated antibodies for drying; available in PBS and TBS with and without T20 Blocker BSA Blocking Buffers Purified bovine serum albumin in PBS or TBS Blocker Casein Blocking Buffers Purified casein in PBS or TBS Blocker BLOTTO Blocking Buffer Non-fat dry milk proteins in TBS SEA BLOCK Blocking Buffer Steelhead salmon serum Protein-Free Blocking Buffers Proprietary non-protein blocking compound; available in PBS and TBS with and without T To order or for technical support, contact your local office or distributor. See pages -2, 3.

23 Western Blotting Blocking Buffers Protein-Free Blocking Buffers Remove background from your research. Thermo Scientific Protein-Free Blocking Buffers provide effective blocking for membrane-based (Western blotting) and plate-based (ELISA) detection methods, resulting in extremely low background. Because these buffers do not contain protein of any kind, they are not susceptible to the types of cross-reactivity and nonspecific binding problems that frequently plague the use of typical protein-based blockers. Protein-Free Blocking Buffers not only enable experiments to proceed when no other blocking agent is effective, they perform so well that they are worth using more generally from the start. Traditional blocking buffers contain proteins that can cross-react with a system, resulting in high background and reduced signal. Protein-Free Blocking Buffers eliminate or minimize cross-reactivity associated with protein-based blocking buffers in ELISA, Western blotting, arrays and other immunodetection applications. Protein-free eliminate or minimize cross-reactivity associated with protein-based blocking buffers Compatible with multiple detection systems can be used in Western blots, ELISA or arrays; do not interfere with avidin-biotin systems High signal-to-background for optimal sensitivity 1X formulation ready to use Available with 0.05% Tween*-20 detergent already added saves time and money 1-Minute Film Exposure 1-Minute Film Exposure Protein-Free Blocking Buffer Non-Animal Protein Blocker X Non-Animal Protein Blocker Y Nitrocellulose PVDF Nitrocellulose PVDF Nitrocellulose PVDF Thermo Scientific Protein-Free Blocking Buffer efficiently blocks Western blotting membranes. Jurkat apoptotic lysate (Lane µg, Lane µg) was separated in 4-20% Tris-glycine gels and transferred to nitrocellulose or PVDF membranes. The membranes were blocked for 1 hour at RT with the indicated blocking buffer, probed with mouse anti-parp (0.25 µg/ml) followed by goat anti-mouse HRP (4 ng/ml) and detected by Thermo Scientific SuperSignal West Dura Chemiluminescent Substrate. Background Signal Non-Animal Protein Blocker X Thermo Scientific Protein-Free Blocking Buffers exhibit less background than other blocking buffers in Thermo Scientific SearchLight Multiplex Arrays. SearchLight* Arrays were created by spotting up to 12 cytokine capture antibodies per well. The plates were blocked with the indicated blocking buffers and the background for each well determined. Error bars represent the standard deviation for triplicate microplate wells. Non-Animal Protein Blocker Y Protein-Free (TBS) Blocking Buffer Proprietary formulation in Tris-buffered saline at ph 7.4 with Kathon* Antimicrobial Agent Protein-Free T20 (TBS) Blocking Buffer Proprietary formulation in Tris-buffered saline at ph 7.4 with 0.05% Tween-20 and Kathon Antimicrobial Agent Protein-Free (PBS) Blocking Buffer Proprietary formulation in phosphate-buffered saline at ph 7.4 with Kathon Antimicrobial Agent Protein-Free T20 (PBS) Blocking Buffer Proprietary formulation in phosphate-buffered saline at ph 7.4 with 0.05% Tween-20 and Kathon Antimicrobial Agent. Protein-Free Blocker 1 L 1 L 1 L 1 L Western Blotting Technical Handbook Western Blotting Handbook and Troubleshooting Guide Featuring Thermo Scientific SuperSignal Substrates and Pierce Western Blotting Accessories The Western Blotting Handbook and Troubleshooting Guide is a 75-page booklet that details each step of the Western blotting process with technical information and products for transfer, blocking, washing, antibodies, substrates, film and stripping buffer. You will want to keep this booklet close at hand because it also includes protocols, references and a troubleshooting guide. To download a copy or view a pdf online, visit To request a printed copy, visit the same website or contact your local office or distributor. Additional dry ice and/or freight charges Additional hazardous handling charge *Trademark, see Trademark Index To order or for technical support, contact your local office or distributor. See pages -2,

24 Western Blotting Blocking Buffers StartingBlock Blocking Buffers Takes the confusion out of choosing a blocker for new experiments. Nitrocellulose 30 minutes PVDF 30 minutes Although no one blocking buffer is ideal for every system, you may never find a closer candidate for that claim than Thermo Scientific StartingBlock Blocking Buffer. Our scientists have yet to encounter an antibody combination or other protein probing system that is not compatible with StartingBlock* Blocking Buffer. Begin with this buffer and you may never look back again! 1A 1B 1C 1D Thermo Scientific StartingBlock Blocking Buffer performance after stripping and reprobing. Nitrocellulose vs. PVDF when probed for the transferrin receptor (CD71), detected with Thermo Scientific SuperSignal West Dura Chemiluminescent Substrate and exposed to film for 30 minutes and 24 hours. Very little background occurs with either membrane or exposure time, indicating exceptional blocking performance. SuperBlock Blocking Buffers Biotin- and serum protein-free blocker. Nitrocellulose 24 hours PVDF 24 hours This immensely popular singleprotein blocking buffer is extremely versatile. Thermo Scientific SuperBlock Buffer blocks ELISA plates and membranes quickly and efficiently, resulting in high signal:noise ratios. The unique formulation stabilizes coated and pre-blocked plates for long-term dry storage. Fast block ELISA plates in ~2 minutes and membranes in < 10 minutes High signal:noise ratio biotin- and serum protein-free formulation Stabilizing formula blocked plates can be stored dry for up to 12 months Convenient formats available in PBS and TBS, with and without Tween*-20 detergent Dry-blend TBS format delivers economy and convenience room temperature storage; pouches take up minimal shelf space and each pack reconstitutes with water to yield 200 ml of SuperBlock* Blocking Buffer in TBS Compatible with many detection systems Western blots, ELISAs and IHC with antibody or avidin/biotin probes (blocker is serumand biotin-free) Short blocking times less than 15 minutes for Westerns, almost instantaneous for ELISAs Strip and reprobe without reblocking blots stay blocked when Thermo Scientific Restore Stripping Buffer (Product # 21059) is used High signal:noise ratio biotin- and serum protein-free formulation Convenient formats available in PBS and TBS, with and without Tween*-20 detergent Reference Lorenzon, N.M., et al. (2004). J. Biol. Chem. 279, StartingBlock (PBS) Blocking Buffer 1 L A protein-based blocker formulation in phosphatebuffered saline (ph 7.5) for use in Western blotting and ELISA applications StartingBlock (TBS) Blocking Buffer 1 L A protein-based blocker formulation in Tris-buffered saline (ph 7.5) for use in Western blotting and ELISA applications StartingBlock T20 (PBS) Blocking Buffer 1 L A protein-based blocker formulation in phosphatebuffered saline at ph 7.5 with 0.05% Tween-20 and Kathon* Antimicrobial Agent StartingBlock T20 (TBS) Blocking Buffer A protein-based blocker formulation in Tris-buffered saline at ph 7.5 with 0.05% Tween-20 and Kathon Antimicrobial Agent. 1 L Ikeda, K., et al. (2003). J. Biol. Chem. 278, Leclerc, G.J. and Barredo, J.C. (2001). Clin. Cancer Res. 7, Subbarayan, V., et al. (2001). Cancer Res. 1, Walters, R.W., et al. (2002). Cell 100, SuperBlock (PBS) Blocking Buffer 1 L SuperBlock (TBS) Blocking Buffer 1 L 3751 SuperBlock T20 (PBS) Blocking Buffer 1 L 3753 SuperBlock T20 (TBS) Blocking Buffer 1 L SuperBlock (PBS) Blocking Buffer Blotting 1 L SuperBlock (TBS) Blocking Buffer Blotting 1 L SuperBlock (TBS) Blocking Buffer Dry Blend Blocking Buffer Each pack yields 200 ml when reconstituted. 5 pack Formulated for precipitating enzyme substrates. Added ingredient to keep ppt from flaking. Not recommended for chemiluminescent substrates. 150 To order or for technical support, contact your local office or distributor. See pages -2, 3.

25 Western Blotting Blocking Buffers SEA BLOCK Blocking Buffer No mammalian proteins, reducing the risk of nonspecific interaction. Thermo Scientific SEA BLOCK Blocking Buffer contains steelhead salmon serum that does not interact substantially with mammalian antibodies, thus resulting in little or no background. Functions as a universal blocker Offers reduced background Can be diluted up to 1:10 with buffer Hypolite, J.A., et al. (2001.) Am J Physiol Cell Physiol. 280, C Wang, L., et al. (2002). J. Clin. Invest. 110, SEA BLOCK Blocking Buffer 500 ml Blocker Casein Ready-to-use solution (1% w/v) of Hammarsten Grade casein for blocking nonspecific sites. Preformulated for ease of use Use when skim milk demonstrates high background problems Thimerosal-free formulation Blocker BLOTTO Ready-to-use blocking buffers made of nonfat dry milk. Preformulated for ease of use Anti-foaming agent added Available in TBS Buffer Thimerosal-free formulation Nemzek, J.A., et al. (2000). Am J Physiol Lung Cell Mol Physiol. 278, L Stuyver, L.J., et al. (2003). Antimicrob. Agents Chemother. 47, Wolfman, J.C., et al. (2002). Mol. Cell. Biol. 22, Blocker Casein in TBS 1% (w/v) Casein Hammarsten Grade in TBS, Kathon* as preservative, ph Blocker Casein in PBS 1% (w/v) Casein Hammarsten Grade in PBS, Kathon as preservative, ph 7.4 Abrams, E.T., et al. (2003). J. Immunol. 170, Goretzki, L., et al. (2000). J. Biol. Chem. 275, Blocker BLOTTO in TBS 5% (w/v) Nonfat powdered milk in TBS, 0.01% Anti-foam A, Kathon* as preservative, ph L 1 L 1 L Blocker BSA For all blocking applications. 10% solutions of high-quality bovine serum albumin Concentrated formulation saves storage space No waiting for powder to dissolve with this ready-to-dilute liquid concentrate Blocker BSA in PBS (10X) 200 ml Blocker BSA in TBS (10X) 125 ml Related Products Page Normal Goat Serum Normal Horse Serum Normal Human Serum Normal Mouse Serum Normal Rabbit Serum Normal Rat Serum Normal Swine Serum Mouse IgG, Fc Fragment 187 Additional dry ice and/or freight charges Additional hazardous handling charge *Trademark, see Trademark Index To order or for technical support, contact your local office or distributor. See pages -2,

26 Western Blotting Buffers Phosphate Buffered Saline (PBS) Great wash buffer for Western blots! Thermo Scientific Pierce 20X Concentrated Buffers are ready to use without having to reconstitute with ultrapure water. Pierce* Concentrated Buffers are designed for use in dialysis, crosslinking, enzyme assays, ELISAs, immunohistochemistry, protein platecoating, biotinylation and other applications. Keep our concentrated buffers close at hand to cover your lab s research needs. Highlights 1X PBS is 0.1 M sodium phosphate, 0.15 M sodium chloride, ph Each dry-blend Thermo Scientific BupH Pack yields 500 ml of 1X PBS when dissolved in 500 ml water 20X formulations offered with and without Tween*-20 detergent BupH Phosphate Buffered Saline Packs 40 pack X Phosphate Buffered Saline 500 ml X PBS Tween-20 (contains 0.05% Tween-20 at 1X) 500 ml Tris Buffered Saline (TBS) Great wash buffer for Western blots! 1X TBS is 25 mm Tris, 0.15 M sodium chloride, ph Each dry-blend Thermo Scientific BupH Pack yields 500 ml of 1X TBS when dissolved in 500 ml water 20X formulations offered with and without Tween*-20 detergent Surfact-Amps 20 Purified Detergent Solution Specially purified form of Tween-20. Thermo Scientific Surfact-Amps Purified Detergent provides unsurpassed purity, quality and stability. Can be added to blocking buffers to reduce background Can be added to PBS or TBS wash buffers to improve performance Guaranteed < 1 milliequivalent of peroxides and carbonyl in a 10% solution Enhances signal-to-background ratio Roti, R.C., et al. (2002). J. Biol. Chem. 277, Singh, R.R., et al. (2003). J. Immunol. 170, BupH Tris Buffered Saline Packs 10 pack 2837 BupH Tris Buffered Saline Packs 40 pack X Tris Buffered Saline 500 ml X TBS Tween-20 (contains 0.05% Tween-20 at 1X) 500 ml Reference Ebong, S.J., et al. (2001). Infect. Immun. 9, Surfact-Amps 20 x 10 ml Tris-Glycine Transfer Buffer A ready-to-use transfer buffer. This is the most common running buffer for electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose or PVDF membranes for subsequent Western blotting detection. The 1X formulation is 25 mm Tris, 192 mm glycine, ph 8, which is typically prepared in 20% methanol. 1X Tris-glycine buffer is 25 mm Tris, 192 mm glycine, ph 8 and is usually prepared in 20% methanol Each dry-blend Thermo Scientific BupH Pack yields 500 ml of 1X TBS when dissolved in 400 ml water and 100 ml methanol 10X formulation makes 1X buffer with ph BupH Tris-Glycine Transfer Buffer Packs 40 pack X Tris-Glycine Buffer 1 L 152 To order or for technical support, contact your local office or distributor. See pages -2, 3.

27 Western Blotting Chemiluminescent Substrates Chemiluminescent Substrates for Western Blotting When energy in the form of light is released from a substance because of a chemical reaction, the process is called chemiluminescence. Luminol is one of the most widely used chemiluminescent reagents, and its oxidation by peroxide results in creation of an excited state product called 3-aminophthalate. This product decays to a lower energy state by releasing photons of light. Enhanced chemiluminescence (ECL) is made possible with proprietary additives that greatly increase the intensity and duration (stability) of light emitted by the luminol reaction. Chemiluminescent substrates have steadily gained in popularity because they offer several advantages over other detection methods. These advantages have allowed chemiluminescence to become the detection method of choice in most protein laboratories. Using chemiluminescence allows multiple exposures to obtain the best image. The detection reagents can be removed and the entire blot reprobed to visualize another protein or to optimize detection of the first protein. A large linear response range allows detection and quantitation for a large range of protein concentrations. Most importantly, chemiluminescence yields the greatest sensitivity of any available detection method. Using HRP as the enzyme label and Thermo Scientific SuperSignal West Femto Chemiluminescent Substrate (Product # 34095), detection limits as low as 1 femtogram are possible because the enhancers in this substrate greatly intensify the emitted light and extend the signal duration. Chemiluminescent substrates differ from other substrates in that the light detected is a transient product of the reaction that is only present while the enzyme-substrate reaction is occurring. This is in contrast to substrates that produce a stable, colored product; these colored Comparison of Thermo Scientific Chemiluminescent Substrates. precipitates remain on the membrane after the enzyme-substrate reaction has terminated. On a chemiluminescent Western blot, the substrate is the limiting reagent in the reaction; as it is exhausted, light production decreases and eventually ceases. A well-optimized procedure using the proper antibody dilutions will produce a stable output of light for several hours, allowing consistent and sensitive detection of proteins. When the antibody is not diluted sufficiently, a stable output of light will never be achieved. Too much enzyme in the system will rapidly oxidize the substrate and terminate the signal. H 2 O HRP Chemiluminescence. Luminol is oxidized in the presence of HRP and hydrogen peroxide to form an excited state product (3-aminophthalate). The 3-aminophthalate emits light at 425 nm as it decays to the ground state. Advantages of enhanced chemiluminescence. Sensitive Fast Stable Hard-copy Results Film Results NH 2 O O NH NH Ability to Reprobe the Blot Large Linear Response Quantitative NH 2 O O O O * NH nm Intense signal with low background Requires less antigen and antibody Rapid substrate processing of blot Signal generated within seconds Unlike radioisotopes, the shelf life is long Store at 4 C Results are captured on X-ray film No fading or tearing of brittle membrane over time Permanent record Signal remains glowing for an extended period of time Ability to place blot against film at various times Ability to optimize the developing method Nonisotopic probes can be stripped off membrane Immunodetection can be repeated Can detect a large range of protein concentrations Can scan the X-ray film using a reflectance densitometer or an imaging device, (e.g., CCD camera) Pierce ECL Substrate SuperSignal West Pico Chemiluminescent Substrate SuperSignal West Dura Extended Duration Substrate Primary Benefit The same signal intensity at Better signal and lower price Extended signal duration is ideal half the price of competing than competing products for use with imaging equipment ECL Substrates Lower Detection Limit Low-microgram (10 - ) Low-picogram (10-12 ) Mid-femtogram (10-14 ) High-picomoles (10-11 ) Mid-attomole (10-17 ) High-zeptomole (10-19 ) Signal Duration 30 Minutes-2 Hours -8 Hours 24 Hours 8 Hours Suggested Antibody Dilutions Primary: 1:100-1:5,000 Secondary: 1:1,000-1:15,000 Room Temperature (RT) Working Solution Stability Primary: 1/1,000-1/5,000 Secondary: 1/20,000-1/100,000 Primary: 1/1,000-1/50,000 Secondary: 1/50,000-1/250,000 1 Hour 24 Hours 24 Hours 8 Hours O O O O SuperSignal West Femto Maximum Sensitivity Substrate The most sensitive chemiluminescent substrate for HRP detection available Low-femtogram (10-15 ) Mid-zeptomole (10-20 ) Primary: 1/5,000-1/100,000 Secondary: 1/100,000-1/500,000 Stock Solution Shelf Life 1 year at 4 C 1 year at RT 1 year at RT 1 year at 4 C or months at RT Lower detection limits were determined using Streptavidin-HRP or Biotinylated-HRP as the ligand. Please follow recommended antibody dilutions. SuperSignal Substrates are much more sensitive than other substrates, so it is critical that you follow these guidelines. Failure to do so could result in unsatisfactory results. Dilutions are from a 1 mg/ml stock solution. Additional dry ice and/or freight charges Additional hazardous handling charge *Trademark, see Trademark Index To order or for technical support, contact your local office or distributor. See pages -2,

28 Western Blotting Chemiluminescent Substrates Pierce ECL Western Blotting Substrate A reliable ECL formulation without inflated prices. Paying twice what you should for an enhanced chemiluminescent (ECL) substrate? For researchers interested in a quality product at a fair price, there is another option available. Thermo Scientific Pierce ECL Western Blotting Substrate is an entry-level Western blotting substrate that is value-priced. If you are currently using a needlessly expensive ECL substrate, you can switch to our ECL Western Blotting Substrate without any optimization. Simply switch out the substrates and save a bundle. Half the cost of other ECL Substrates low overhead and a commitment to customer value enables us to make this product available to you for half of the cost other companies charge (these claims are based on the 2008 U.S. list prices) No optimization required you can switch to our ECL substrate without the need for optimization or protocol changes A name you can rely on we put our strong technical support and reputation behind this product Thermo Scientific Pierce ECL Reagent GE Healthcare (Amersham) ECL Reagent 1-minute exposure 1-minute exposure Relative Intensity Units 1,800,000 1,00,000 1,400,000 1,200,000 1,000, ,000 00, , ,000 0 Protein per well GE Healthcare ECL Thermo Scientific Pierce ECL 4 µg 2 µg 1 µg 0.5 µg 0.25 µg Signal intensity of Thermo Scientific Pierce ECL Substrate is comparable to GE Healthcare ECL Substrate. HeLa cell lysate was separated by SDS-PAGE and transferred to nitrocellulose membrane to detect β-actin. The signal was detected and analyzed using the Kodak 1D Imaging Station. Error bars represent ±20% difference in relative intensity units. Recombinant Bovine TNF-α on Nitrocellulose β-galactosidase from E. coli on PVDF β-actin from HeLa Cell Lysate on Nitrocellulose Protein per well (20 kda) Protein per well ( kda) Protein per well (40-50 kda) MW Marker (pg) MW Marker (ng) MW Marker (mg) 5-minute exposure 5-minute exposure 1.5-minute exposure 1.5-minute exposure Thermo Scientific Pierce ECL Substrate performs identically to GE ECL Substrate for a variety of conditions. Columns represent three different Western blot experiments performed identically except for the substrate product used. Working solutions of the two brands of substrate were prepared according to the manufacturer s instructions and added to the membranes for 1 minute. Regardless of protein size, type and quantity, or the film exposure time used, Pierce ECL Substrate performed like the more expensive GE Healthcare Product Pierce ECL Western Blotting Substrate Sufficient substrate for 4,000 cm 2 of membrane Pierce ECL Western Blotting Substrate Sufficient substrate for 2,000 cm 2 of membrane Pierce ECL Western Blotting Substrate Sufficient substrate for 400 cm 2 of membrane. 500 ml kit 250 ml kit 50 ml kit 154 To order or for technical support, contact your local office or distributor. See pages -2, 3.

29 Western Blotting Chemiluminescent Substrates SuperSignal West Pico Chemiluminescent Substrate Twice as much signal for less than the GE ECL System. 1 minute exposure 50 ng 3 pg 5 minute exposure 50 ng 3 pg Thermo Scientific SuperSignal West Pico Substrate GE Healthcare ECL System Thermo Scientific SuperSignal West Pico is more sensitive than GE ECL Substrate. Recombinant mouse IL-2 was serially diluted ( ng) and electrophoresis was performed. The gels were transferred to nitrocellulose membranes, blocked and incubated with a 1 µg/ml dilution of Rat Anti-Mouse IL-2. After washing, This SuperSignal Substrate System yields longer light emission and at least two times the intensity of other entry-level luminol-based systems while maintaining sensitivity. All this for about two-thirds the price of comparable products. Economy costs less per milliliter than other similarly sensitive chemiluminescent substrate available Picogram sensitivity highly sensitive for the rapid development of a wide range of protein levels Longer light emission strong light emission over a working day allows you to make several exposures High intensity signal is twice as intense as other luminol-based systems Excellent stability 24-hour plus working solution stability; kit is stable for at least one year at room temperature Saves antibody requires more dilute primary and secondary antibodies so you use less per blot Net Relative Intensity 500, , , , ,000 0 Thermo Scientific SuperSignal West Pico Substrate GE Healthcare ECL System Enhanced light emission kinetics: Thermo Scientific SuperSignal Substrate vs. GE ECL System. Net relative intensity six hours after incubation is much greater for SuperSignal West Pico Substrate than for the GE ECL System. the membranes were incubated with 20 ng/ml dilutions of HRP-conjugated Goat Anti-Rat antibody. The membranes were washed again and then incubated with substrate that was prepared according to the manufacturers instructions. Blots were exposed to film for one- and five-minute exposures. Butchbach, M., et al. (2004). J. Biol. Chem. 279, Ju, T., et al. (2002). J. Biol. Chem. 277, Kagan, A., et al. (2000). J. Biol. Chem. 275, Messenger, M.M., et al. (2002). J. Biol. Chem. 277, Sorci, G., et al. (2003). Mol. Cell. Biol. 23, SuperSignal West Pico Chemiluminescent Substrate Sufficient substrate for 10,000 cm 2 of blotting. Includes: Luminol/Enhancer Stable Peroxide Buffer SuperSignal West Pico Substrate Sufficient substrate for 5,000 cm 2 of blotting. Includes: Luminol/Enhancer Stable Peroxide Buffer SuperSignal West Pico Substrate Sufficient substrate for 1,000 cm 2 of blotting. Includes: Luminol/Enhancer Stable Peroxide Buffer SuperSignal West Pico Substrate Trial Kit Sufficient substrate for 500 cm 2 of blotting. Includes: Luminol/Enhancer Stable Peroxide Buffer Standard Detection Kits HRP-conjugated secondary antibody SuperSignal West Pico Substrate (500 ml) SuperSignal West Pico Mouse IgG Detection Kit SuperSignal West Pico Rabbit IgG Detection Kit Complete Detection Kits HRP-conjugated secondary antibody SuperBlock Blocking Buffer (1 L) Tris-buffered Saline (TBS), 4 packs (500 ml each) SuperSignal West Pico Substrate (500 ml) SuperSignal West Pico Complete Mouse IgG Detection Kit SuperSignal West Pico Complete Rabbit IgG Detection Kit Patent, see Patent Index. 1 L 500 ml 500 ml 500 ml 250 ml 250 ml 100 ml 50 ml 50 ml 50 ml 25 ml 25 ml Kit Kit Kit Kit Additional dry ice and/or freight charges Additional hazardous handling charge *Trademark, see Trademark Index To order or for technical support, contact your local office or distributor. See pages -2,

30 Western Blotting Chemiluminescent Substrates SuperSignal West Dura Extended Duration Substrate Specially formulated for use with cooled-ccd cameras. 1 minute exposure 50 ng 3 pg Cooled CCD Camera (30 and 15 minutes) 50 ng 3 pg Thermo Scientific SuperSignal West Dura Substrate GE Healthcare (Amersham) ECL Plus System Thermo Scientific SuperSignal West Dura provides better sensitivity and less background than GE Healthcare ECL Plus Substrate. Recombinant mouse IL-2 was serially diluted ( ng) and electrophoresed. The gel to be used for SuperSignal West Dura Substrate was transferred to nitrocellulose membrane and the gel to be used for ECL Plus Substrate was transferred to PVDF membrane (manufacturer s recommendation). The membranes were blocked and then incubated with a 1 µg/ml dilution of the primary antibody, rat anti-mouse IL-2. After washing, the membranes Thermo Scientific SuperSignal West Dura Extended Duration Substrate for HRP is optimized for high sensitivity and long signal duration, making it ideal for cooled-ccd camera detection systems. Unlike other substrates, whose signal declines to useless levels in 30-0 minutes, the signal produced with SuperSignal* West Dura Substrate is stable for several hours, allowing multiple film or camera exposures to be performed. 24-hour light emission 10 times longer than with other enhanced chemiluminescent substrates for HRP; make multiple exposures for publication-quality blots Great sensitivity with femtogram-level detection, see bands you ve never been able to see before Save your antibody requires very dilute antibody concentrations, allowing you to perform times more blots than with other chemiluminescent substrates Intense immediate stable signal generation provides easy detection on film or compatible imaging system Working solution is stable for at least 24 hours the kit itself is stable for at least one year with ambient shipping conditions 24-hour signal duration and high intensity. Elapsed Time After Substrate Incubation Exposure Period Dot Blot Image 5 minutes 5 minutes were incubated with the secondary antibody, HRP-conjugated goat anti-rat IgG. The membranes were washed again and then incubated with substrates that were prepared according to the manufacturer s instructions. Each membrane was exposed to X-ray film for 5 minutes. The SuperSignal West Dura Substrate membrane was exposed to the ChemiImager 4000 for 30 minutes and the ECL Plus Blot was exposed for 15 minutes. (The exposure of the ECL Plus Blot was not extended to 30 minutes due to the high background that had already accumulated at 15 minutes.) Gangalum, R.K. et al. (2004). J. Biol. Chem. 279, MacInnis, B.L. and Campenot, R.B. (2004). Science 295, Sato, S., et al. (2003). J. Biol. Chem. 278, Tokumaru, H., et al. (2001). Cell 104, SuperSignal West Dura Extended Duration Substrate Sufficient for 2,000 cm 2 of blotting membrane. Includes: Luminol/Enhancer Stable Peroxide Buffer SuperSignal West Dura Extended Duration Substrate Sufficient for 1,000 cm 2 of blotting membrane. Includes: Luminol/Enhancer Stable Peroxide Buffer SuperSignal West Dura Extended Duration Substrate Trial Kit Sufficient for 200 cm 2 of blotting membrane. Includes: Luminol/Enhancer Stable Peroxide Buffer Patent, see Patent Index. 200 ml 100 ml 100 ml 100 ml 50 ml 50 ml 20 ml 10 ml 10 ml Related Products Page # 251 Chemiluminescent Prestained Peroxidase- Labeled Protein Molecular Weight Marker Mix Restore Western Blot Stripping Buffer Pierce Background Eliminator for Film 12 1 hour 5 minutes 8 hours 5 minutes 24 hours 1 hour These images illustrate SuperSignal West Dura Substrate s more than 24-hour signal duration and how well the signal is captured using a cooled CCD camera. Using the Alpha Innotech Corporation ChemiImager* 4000, images of a 37 pg HRP dot blot incubated for 5 minutes with SuperSignal West Dura Substrate were taken at the indicated time after incubation with the substrate and with the indicated exposure periods. 15 To order or for technical support, contact your local office or distributor. See pages -2, 3.

31 Western Blotting Chemiluminescent Substrates SuperSignal West Femto Maximum Sensitivity Substrate Detect protein amounts you never could before! Thermo Scientific SuperSignal West Femto Substrate is the most sensitive chemiluminescent substrate system for Western blotting with horseradish peroxidase (HRP) enzyme. The substrate kit enables detection of lowfemtogram (that's mid-zeptomole!) amounts of protein deposited on nitrocellulose or PVDF membrane and probed with appropriate primary and secondary antibodies. When combined with optimization of antibody concentrations and blocking buffers, SuperSignal* West Femto Substrate can make experiments successful that were never before possible because the target proteins occur in such small amounts. Sensitive obtain low-femtogram (mid-zeptomole) protein detection Conserve costly antibodies suggested antibody dilutions (from 1 mg/ml stock): Primary: 1/5,000-1/100,000 Secondary: 1/100,000-1/500,000 Intense signal easy to capture an image on film or using an imager system Excellent signal duration 8 hours Stable reagent 1 year at 4 C or months at RT Quantitative measurable signal over two orders of magnitude fg 10 fg 1 fg True femtogram detection of IκB using Thermo Scientific SuperSignal West Femto Maximum Sensitivity Substrate. Serially diluted samples from 100 to 1 fg were run on 4-20% Thermo Scientific Precise Precast Gels. The protein was then transferred to PVDF membrane and blocked with Thermo Scientific StartingBlock Blocking Buffer for 1 hour at room temperature (RT). The blot was incubated in Rabbit Anti-IκBa (1 mg/ml) at 1:1,000 dilution overnight at 4 C, followed by incubation in Goat Anti-Rabbit HRP (1 mg/ml) at 1:200,000 dilution for 1 hour at RT. The membrane was exposed to Thermo Scientific CL-XPosure Film for 1 minute. Thermo Scientific SuperSignal West Femto Substrate GE Healthcare ECL Plus Substrate Thermo Scientific SuperSignal West Femto is more sensitive than GE Healthcare ECL Plus Substrate. Two-fold serial dilutions of mouse IL-2 were detected with both substrates. The primary antibody dilution was 1/2,000 and the secondary antibody dilution was 1/300,000. Both antibodies had a 1 mg/ml starting concentration. 1. Feissner, R., et al. (2003). Anal. Biochem. 315, Adilakshmi, T. and Laine, R.O. (2002). J. Biol. Chem. 277, Conti, L.R., et al. (2001). J. Biol. Chem. 27, Guo, Y., et al. (2001). J. Biol. Chem. 27, SuperSignal West Femto Maximum Sensitivity Substrate Sufficient substrate for 2,000 cm 2 of blotting membrane. Includes: Luminol/Enhancer Solution Stable Peroxide Solution SuperSignal West Femto Maximum Sensitivity Substrate Sufficient substrate for 1,000 cm 2 of blotting membrane. Includes: Luminol/Enhancer Solution Stable Peroxide Solution SuperSignal West Femto Maximum Sensitivity Substrate Trial Kit Sufficient substrate for 200 cm 2 of blotting membrane. Includes: Luminol/Enhancer Solution Stable Peroxide Solution Patent, see Patent Index. 200 ml 100 ml 100 ml 100 ml 50 ml 50 ml 20 ml 10 ml 10 ml Related Products Page # Restore Western Blot Stripping Buffer Pierce Background Eliminator for Film 12 Pierce O-GlcNAc Western Blot Detection Kit Complete, highly specific kit for detection of O-GlcNAc protein modifications. The Thermo Scientific Pierce O-GlcNAc Western Blot Detection Kit contains the most highly specific mouse monoclonal antibody available for the detection of O-GlcNAc post-translational modification, as well as the cell lysis reagents, secondary antibody and chemiluminescent detection reagents needed to perform an entire detection experiment. Reaction of the primary antibody (clone CTD 110.) is confined to the β-o-linked serine or threonine GlcNAc modification; there is no crossreactivity with the α-o-glcnac linkage, the α/β-o-galnac modification or other N-linked oligosaccharides Pierce O-GlcNAc Western Blot Detection Kit Sufficient material to develop up to 10 mini-blots. Includes: M-PER Reagent Dilution/Blocking Buffer (10X) Phosphate Buffered Saline Surfact-Amps 20 Positive Control Anti O-GlcNAc Primary Antibody Goat Anti-Mouse IgM(µ)-HRP SuperSignal West Dura Substrate Kit 25 ml 2 x 50 ml 17 packs 3 x 10 ml 100 ng 100 µl 75 µg 100 ml Additional dry ice and/or freight charges Additional hazardous handling charge *Trademark, see Trademark Index To order or for technical support, contact your local office or distributor. See pages -2,

32 Western Blotting Chemiluminescent Substrates Lumi-Phos WB Chemiluminescent Substrate Alkaline phosphatase detection that provides the best of both worlds high sensitivity and low background. Thermo Scientific Lumi-Phos WB Substrate provides highly sensitive chemiluminescent Western blot detection of alkaline phosphatase (AP) enzyme conjugates. The single-component substrate is easy to use and produces less background than other popular chemiluminescent subtrates for AP. The result is a better signal-to-noise ratio and a clearer image. Because signal generation is immediate, there s no need to wait 15 to 30 minutes for a measurable signal. High sensitivity able to detect 1.2 pg (71 attomoles) of the target ligand mouse IL-2 Low background high signal:noise ratios produce clearer blots Inexpensive less expensive than CDP-Star* Substrate (based on 2008 U.S. list prices), and there is no need to purchase additional enhancers for nitrocellulose membranes Long signal duration allows for multiple film exposures Immediate strong signal no more waiting 15 to 30 minutes for the signal to become strong enough to detect Ready to use no mixing required with this one-component system DyLight 80/800 Near Infrared Western Blotting Kit Compatible with LI-COR Odyssey* and other fluorescent imaging systems. Near infrared fluorescent detection of two different targets on a single Western blot is easy to perform with the Thermo Scientific DyLight 80/800 Western Blotting Kit. The kit provides highly optimized reagents and a convenient format to save you the time and frustration of having to evaluate reagents for compatibility with fluorescent Western blotting. Each kit contains sufficient reagents for 10 Western blots and includes DyLight* 80/800 Infrared Protein Molecular Weight Markers and secondary antibodies conjugated to DyLight 80 and 800 Fluorescent Dyes. Tubulin TNFα 1 µg 0.5 µg 0.25 µg µg 0.03 µg.25 µg 12.5 µg 25 µg 50 µg 20 ng 1.2 pg Thermo Scientific Lumi-Phos Substrate provides high sensitivity and low background. Two-fold serial dilutions of recombinant mouse IL-2 were separated on a 4%-20% SDS polyacrylamide gel, then transferred to nitrocellulose membrane. The membranes were subsequently probed with purified rat anti-mouse IL-2 (1 µg/ml), followed by goat anti-rat IgG-AP conjugate (200 ng/ml). The membranes were washed and then incubated in Lumi-Phos WB Substrate for five minutes prior to film exposure. Bruinsma, P., et al. (2004). J. Biol. Chem. 279, Dhingra, A., et al. (2004). Proc. Nat. Acad. Sci. USA 101, Tikhonov, I., et al. (2003). J. Virol. 77, Lumi-Phos* WB Chemiluminescent Substrate 100 ml DyLight 80 excitation/emission maxima 92/712 nm DyLight 800 excitation/emission maxima 777/790 nm Optimized format provides low background and high signal intensity Convenient saves time and money associated with optimizing fluorescent Western blots DyLight 80/800 Western Blotting Kit Sufficient reagents for 10 Western blots. Includes: DyLight 80 Goat Anti-Mouse IgG (H+L) DyLight 800 Goat Anti-Rabbit IgG (H+L) DyLight 80/800 Infrared Protein MW Markers Wash Buffer (30X) SEA BLOCK Blocking Buffer Low-Fluorescence PVDF Transfer Membrane Kit 150 µl 150 µl 30 µl 200 ml 500 ml 10 ea. Related Products Page # 2280 Low-Fluorescence PVDF Transfer Membrane SEA BLOCK Blocking Buffer DyLight 80/800 Infrared Protein 109 Molecular Weight Markers The Thermo Scientific DyLight 80/800 Western Blotting Kit provides low background and high signal in two-color Western blot detection. Proteins were separated in a 4-20% Thermo Scientific Precise Protein Gel and transferred to a low-fluorescence PVDF membrane. The membrane was blocked overnight in Thermo Scientific SEA BLOCK Blocking Buffer and target proteins were detected according to the manufacturer s protocol. Membranes were imaged with the LI-COR Odyssey Infrared Imaging System. Tubulin was detected from the indicated quantity of HeLa cell lysate. Purified TNFα was detected at the indicated quantity. 158 To order or for technical support, contact your local office or distributor. See pages -2, 3.

33 Western Blotting Chemiluminescent Substrates DyLight 549/49 Western Blotting Kit Fluorescent Western blotting made easy! The fluorescent detection of two different targets on a single Western blot is easy to perform with the Thermo Scientific DyLight 549/49 Western Blotting Kit. This highly optimized and convenient format saves you the time and frustration of having to evaluate reagents for compatibility with fluorescent Western blotting (Table). The kit contains sufficient reagents for 10 Western blots and includes DyLight* 549/49 Fluorescent Protein Molecular Weight Markers and secondary antibodies conjugated to DyLight 549 and 49 Fluorescent Dyes. DyLight 549 excitation/emission maxima 52/57 nm DyLight 49 excitation/emission maxima 54/73 nm Optimized format provides low background and high signal intensity Convenient saves time and money associated with optimizing fluorescent Western blots Recommended instruments for in-gel and Western blot detection using Thermo Scientific DyLight Fluors. Company Instrument Excitation (mm) Emission (nm) Thermo Scientific DyLight 549 Dye DyLight Dye performance has been evaluated with this instrument. Compatibility of other instruments is based on manufacturers specifications. Thermo Scientific DyLight 49 Dye Kodak Image Station 2000MM 535/25 00/700 Image Station 535/25 00/ MM Bio-Rad Molecular Imager* FX (FX Pro) 532/35 05/95 Amersham Typhoon /33 580/70 Typhoon /33 580/70 Typhoon /33 580/70 Typhoon /33 580/70 Storm* Not compatible Storm Not compatible Fuji FLA /33 570/75 FLA /33 570/75 FLA /33 570/75 Tubulin TNFα Tubulin TNFα 25 ng 12 ng ng 3 ng 1 ng 25 ng 12 ng ng 3 ng 1 ng 0.5 ng 1 ng 0.5 ng 1 ng 3 ng ng 12 ng 25 ng ECL Plex Western Blotting Kit 3 ng ng 12 ng 25 ng MW Marker Thermo Scientific DyLight 549/49 Western Blotting Kit DyLight 549/49 Western Blotting Kit Sufficient reagents for 10 Western blots. Includes: DyLight 549 Goat Anti-Mouse IgG (H+L) DyLight 49 Goat Anti-Rabbit IgG (H+L) DyLight 549/49 Fluorescent Protein MW Markers Wash Buffer (30X) Blocker BSA in PBS (10X) Low-Fluorescence PVDF Transfer Membrane The Thermo Scientific DyLight 549/49 Western Blotting Kit provides lower background and higher signal in two-color Western blot detection compared to another supplier s fluorescent Western blotting kit. Proteins were separated in 4-20% Thermo Scientific Precise Protein Gels and transferred to low-fluorescence PVDF membrane. The membranes were blocked overnight in 1% BSA and target proteins were detected following manufacturer-recommended protocols. Membranes were imaged with the Typhoon* Kit 50 µl 50 µl 0 µl 200 ml 50 ml 10 ea. Related Products Page # 2280 Low-Fluorescence PVDF Transfer Membrane DyLight 549/49 Fluorescent Protein 109 Molecular Weight Markers MW Marker Western Blotting Technical Handbook Western Blotting Handbook and Troubleshooting Guide Featuring Thermo Scientific SuperSignal Substrates and Pierce Western Blotting Accessories The Western Blotting Handbook and Troubleshooting Guide is a 75-page booklet that details each step of the Western blotting process with technical information and products for transfer, blocking, washing, antibodies, substrates, film and stripping buffer. You will want to keep this booklet close at hand because it also includes protocols, references and a troubleshooting guide. To download a copy or view a pdf online, visit To request a printed copy, visit the same website or contact your local office or distributor. Additional dry ice and/or freight charges Additional hazardous handling charge *Trademark, see Trademark Index To order or for technical support, contact your local office or distributor. See pages -2,

34 Western Blotting In-gel Detection Pierce In-Gel Chemiluminescent Detection Kits Detection of difficult-to-transfer proteins. Thermo Scientific Pierce In-Gel Chemiluminescent Detection Kits comprise an innovative variation on traditional Western blotting by making it possible to probe and detect proteins directly in the gel. This method circumvents the transfer and blocking steps entirely, enabling immunoblotting techniques to be applied to proteins that cannot be transferred efficiently from a gel to a membrane. No transfer step means no skewing of protein amounts or other artifacts Compatible with stripping and reprobing protocols Compatible with protein staining Sensitivity comparable to typical Western blotting methods Benefits: Many proteins, such as membrane proteins, do not transfer well to membranes; the Pierce In-Gel Detection Method prevents any problems associated with incomplete transfer When transferring to membranes, low MW proteins transfer more efficiently than higher MW proteins, often skewing results Transfer units, buffers, membranes and filter paper are eliminated Procedure can be optimized by stripping and reprobing without running another gel After immunodetection, the gel can be used for total protein staining; there s no need to run two gels The blocking step is omitted because the antibodies bind only specific antigens in the gel Notes: The Pierce In-Gel Technology has been tested successfully with Novex, FMC-BioWhittaker and Bio-Rad Criterion* gels. The Pierce In-Gel Technology does not perform well with Bio-Rad Ready Gel* or Gradipore igel* Gels; studies showed 25 times lower sensitivity and require individual optimization. The recommended gel thickness for use with this kit is mm. When using Pierce In-Gel Technology with homemade gels, the glass plates must be siliconized prior to pouring the gel. Please visit our website to review the protocol and see other tips on optimizing Pierce In-Gel Technology. In-Gel Detection High sensitivity using Thermo Scientific Pierce In-Gel Detection. Pure GFP xhis-tagged and yeast GFP extract were separated by SDS-PAGE. One gel was transferred to nitrocellulose membrane. After transfer, the membrane was blocked overnight in 1% BSA. The other gel was pre-treated with 50% isopropanol. Antigens were detected using a 1:1,000 dilution of polyclonal Anti-Living Color Peptide Antibody, Rabbit (Clontech) and the Pierce In-Gel Chemiluminescent Detection Kit Rabbit (Product # 33500). Signal was detected using Pierce In-Gel Detection Substrate. The lanes on the gel and in the membrane are as follows: Lanes 1, 2 and 3 correspond to 10, 5 and 1 ng pure GFP/xHis-tagged, respectively. Lanes 4 and 5 correspond to E. coli bacterial GFP lysate diluted 1:100 and 1:1,000, respectively. Lanes and 7 correspond to yeast GFP Lysate diluted 1:10 and 1:100, respectively. De Ioannes, P., et al. (2004). J. Biol. Chem. 279, Desai, S., et al. (2001). Anal. Biochem. 297, Desai, S., et al. (2002). Immunodetection of proteins within polyacrylamide gels. Bioluminescence and Chemiluminescence. World Scientific Publishing Co., pp Roberts, K.P., et al. (2002). Biol. Reprod. 7, Wong, W.K.P., et al. (2005). Am. J. Respir. Cell Mol. Biol, 33, Membrane Detection Pierce In-Gel Chemiluminescent Detection Kit Rabbit Sufficient reagents to perform 10 mini-gel detections. Includes: Pierce In-Gel Substrate Stabilized Goat Anti-Rabbit-HRP (10 µg/ml) Dilution Buffer (10X) BupH Pack PBS Buffer 10% Tween*-20 Incubation Colander Pre-cut Cellophane CL-XPosure Film (5" x 7") Pierce In-Gel Chemiluminescent Detection Kit Mouse Sufficient reagents to perform 10 mini-gel detections. Includes same components as Product # except it contains Goat Anti-Mouse-HRP instead of Goat Anti-Rabbit-HRP Pierce In-Gel Chemiluminescent Detection Kit for Biotinylated Antibody Probes Sufficient reagents to perform 10 mini-gel detections. Includes: Streptavidin-HRP Dilution Buffer (10X) BupH Phosphate Buffered Saline 10% Tween-20 Pierce In-Gel Substrate Cellophane Exposure Sheets Kit 110 ml 1 ml 50 ml 17 packs 5 x 10 ml 1 unit 10 sheets 25 sheets Kit Pierce In-Gel Detection Chemiluminescent Substrate 110 ml Incubation Colander 1 unit Patent, see Patent Index. Related Products Page # Biotinylated Protein Interaction Pull-Down Kit Far-Western Biotinylated-Protein 220 Interaction Kit Kit 1 mg 50 ml 17 packs x 10 ml 110 ml 10 pack 10 To order or for technical support, contact your local office or distributor. See pages -2, 3.

35 Western Blotting X-ray Film CL-XPosure Film Save 5-75% on film! Thermo Scientific CL-XPosure Radiography Film is a high-performance, single-emulsion, clear-blue film for documentation of chemiluminescent Western blotting signals and other light-emitting or radioisotopic assay signals. The economically priced film exhibits the same detection sensitivity as other, more expensive, commercially available film brands. The film works very well with Thermo Scientific Pierce ECL and SuperSignal Chemiluminescent Substrates and other chemiluminescent substrate systems. Much lower price than other suppliers products Provides the same detection sensitivity as other commercially available films (Figure) Available in 5 x 7, 8 x 10, 9.5 x 11.8, 14 x 17 or 18 x 24 cm sheets, in packages of 25, 50 or 100 non-interleaved sheets Western Blotting Technical Handbook Western Blotting Handbook and Troubleshooting Guide Featuring Thermo Scientific SuperSignal Substrates and Pierce Western Blotting Accessories The Western Blotting Handbook and Troubleshooting Guide is a 75-page booklet that details each step of the Western blotting process with technical information and products for transfer, blocking, washing, antibodies, substrates, film and stripping buffer. You will want to keep this booklet close at hand because it also includes protocols, references and a troubleshooting guide. To download a copy or view a pdf online, visit To request a printed copy, visit the same website or contact your local office or distributor. Thermo Scientific Kodak* BioMax* Kodak X-Omat* CL-XPosure Film MR-1 Film Blue (XB) Film Thermo Scientific CL-XPosure Film vs. Kodak Film. Three types of X-ray film were tested using identical Western blotting conditions (2 blue, 1 grey). The results showed no appreciable difference between any of these films. The only significant difference is the cost-per-sheet of film. Reference Tikhonov, I., et al. (2003). J. Virol. 77, CL-XPosure Film, 5 x 7 in (13 x 18 cm) 100/pkg CL-XPosure Film, 5 x 7 in (13 x 18 cm) 25/pkg CL-XPosure Film, 7 x 9.5 in (18 x 24 cm) 100/pkg CL-XPosure Film, 8 x 10 in (20 x 25 cm) 100/pkg CL-XPosure Film, 8 x 10 in (20 x 25 cm) 50/pkg CL-XPosure Film, 9.5 x 11.8 in (24 x 30 cm) 100/pkg CL-XPosure Film, 14 x 17 in (35 x 43 cm) 100/pkg. Additional dry ice and/or freight charges Additional hazardous handling charge *Trademark, see Trademark Index To order or for technical support, contact your local office or distributor. See pages -2, 3. 11

36 Western Blotting Background Eliminator Pierce Background Eliminator for Film Easily remove background so you can see your results clearly. High background, shading, overexposed bands and speckling are problems inherent to film exposure. High background and shading can be caused by overexposure, poor use of blocking buffer or inappropriate enzyme-labeled probe or antibody concentration. Overexposed bands are a common occurrence when the enzyme-labeled probe or antibody used is too concentrated or if the film was exposed for too long. Speckling and shading occur when enzyme conjugates form complexes and precipitate on the blot. Thermo Scientific Pierce Background Eliminator can correct all these problems without the need to re-expose your blot to film or re-do the experiment, allowing you to visualize your data within minutes. The product can be used with any brand of newly exposed films or exposed films that have been stored for years. For applications requiring densitometric measurement, the Pierce* Background Eliminator reduces signal evenly over the film so that relative densitometry values are consistent. The procedure is simple. Immerse your exposed film in Pierce Background Eliminator Working Solution, watch for desired image and stop the reaction by rinsing the film in water. The solution works quickly, achieving ideal signal levels in just a few minutes. Reduces signal evenly over the film no altering of results Rapid and trouble-free correction of overexposed, speckled or shaded films Works with any X-ray film, new or old No need for time-consuming re-exposures to find the optimal image No need to re-optimize assay reagents to obtain the optimal image Remove background from any application that uses X-ray film exposures including: Western, Northern and Southern blots that use Thermo Scientific SuperSignal Substrates and the Pierce Chemiluminescent Hybridization and Detection Kit In-gel detection systems Gel-shift assays Ribonuclease protection assays (RPA) Old option: Start over and re-optimize antibody concentration and blocking buffer. A. Overexposed Film: B: Two days later C: Four minutes later Thermo Scientific Pierce Background Eliminator lightens the entire film evenly in four minutes vs. the two days traditional methods require to start over and reoptimize experiment conditions. A431 cell lysate was electrophoresed on a 4-12% Tris-glycine gel and transferred overnight to nitrocellulose. The membrane was blocked with Thermo Scientific SuperBlock Blocking Buffer in PBS (Product # 37515) for 1 hour and incubated with 1.25 ng/ml of HRP-labeled mouse anti-phosphotyrosine (PY20) for 1 hour. After the membrane was washed for 30 minutes, SuperSignal West Dura Substrate was added. The blot was exposed to film for 10 seconds and resulted in a completely black image caused by the antibody cross-reacting with the blocking buffer (A). Using the old option, another gel was prepared to optimize assay conditions. The proteins were transferred overnight and then the membrane was blocked with a 5% dry milk solution for 1 hour. The blot was detected with 2.5 ng/ml of anti-phosphotyrosine (PY20)-HRP and SuperSignal West Dura Substrate. The blot was exposed to film for 10 seconds. This optimization required a two-day procedure (B). Using the new option, the initial dark film (A) was treated with Pierce Background Eliminator to allow the band images to appear in 4 minutes (C). Before Treatment After Treatment New option: Use Thermo Scientific Pierce Background Eliminator. A. B. Thermo Scientific Pierce Background Eliminator erases speckling. Recombinant Human TNFα was electrophoresed on a 4-20% SDS-polyacrylamide gel and transferred to a nitrocellulose membrane. The membrane was blocked and detected with Mouse anti-human TNFα followed by Thermo Scientific Goat anti-mouse-hrp (Product # 31434) and SuperSignal West Dura Substrate (Product # 34075). The blot was exposed to film for 30 seconds, resulting in considerable background speckling (A). The film was then treated with Pierce Background Eliminator for 2 minutes to eliminate the background speckling (B) Pierce Background Eliminator Sufficient reagent to prepare 3 L of working solution. Includes: Reagent A Reagent B Kit 100 ml 100 ml 12 To order or for technical support, contact your local office or distributor. See pages -2, 3.

37 Western Blotting Stripping Buffers Restore Western Blot Stripping Buffer Strip time off your research. Tired of re-running electrophoresis gels and waiting to see your results? Although optimizing assay conditions is the best way to achieve optimum results, re-performing the gel electrophoresis process to test each new primary antibody or antibody concentration is time-consuming and expensive. You can forget about starting over when you use Thermo Scientific Restore Western Blot Stripping Buffer! Saves time no need to re-run gels Saves precious sample re-probe the membrane using the same target sample Provides efficient removal proprietary formulation works better than homemade buffers Gentle formulation does not damage target protein after stripping and re-probing Odor-free no mercaptans means no acrid odors Economical less expensive than other competing stripping buffers Test different primary antibodies There s no need to waste precious sample and re-run a gel to test different primary antibodies. Simply strip the membrane with Restore Stripping Buffer to remove the first primary antibody and set of reagents. It takes only 5-15 minutes, depending on the affinity of the primary antibody. After stripping, re-probe with a new primary antibody (see figure). Antibody #1 Anti-JAK-1 Stripped No Ab-Clean Antibody #2 Anti-Bak Optimize assay conditions Using Thermo Scientific SuperSignal West Substrates, secondary antibody concentrations can be optimized after a single stripping and re-probing cycle (see figure). A chemiluminescent detection system is the most sensitive method to detect any reagent still bound after the stripping procedure. A. B. C. Antibody optimization study. Western blots of Interleukin-2 (diluted ng) were detected using SuperSignal West Pico Chemiluminescent Substrate. The first blot (A) used the primary antibody diluted to 1/1,000 (0.5 µg/ml) of rat anti-mouse IL-2 and the horseradish peroxidase (HRP)-labeled goat anti-rat secondary antibody (Product # 31470) diluted 1/5,000. The same blot was stripped with Restore Western Blot Stripping Buffer (B) for 5 minutes at room temperature and re-probed (C) with the primary antibody at 1/5,000 and the HRP-secondary conjugate at 1:20,000. Thermo Scientific SuperBlock Blocking Buffer was used for blocking. Abulrob, A., et al. (2005). J. Neurochem. 192, Gordon-Thomson, C., et al. (2005). Melanoma Res. 15, Kaufmann, S.H., et al. (1987). Anal. Biochem. 11, Kaufmann, S.H. and Kellner, U. (1998). Erasure of Western blots after autoradiographic or chemiluminescent detection. In Immunochemical Protocols. Ed. Pound, J.D. Humana Press, Totowa, NJ, Schrager, J.A., et al. (2002). J. Biol. Chem. 277, Sorci, G., et al. (2003). Mol. Cell. Biol. 23, Suzuki, E., et al. (2004). Clin. Cancer Res. 10, Restore Western Blot Stripping Buffer 500 ml Sufficient buffer for stripping 25 (8 cm x 10 cm) blots Restore Western Blot Stripping Buffer 30 ml Related Products Page # SuperBlock Blocking Buffer in PBS SuperSignal West Pico 155 Chemiluminescent Substrate SuperSignal West Dura Extended Duration 15 Chemiluminescent Substrate SuperSignal West Femto Maximum Sensitivity Substrate 157 Patent, see Patent Index. Re-probing with different antibodies. Western blots of HeLa cell lysate protein (diluted from 750 ng to 83.3 ng) were detected with SuperSignal West Dura Chemiluminescent Substrate. The first blot used polyclonal rabbit anti-jak-1 primary antibody at 1:2,000 dilution with an HRP-secondary conjugate diluted at 1:350,000. The same blot was stripped for 5 minutes at room temperature in Restore Western Blot Stripping Buffer and then re-probed with purified mouse anti-human Bak monoclonal primary antibody at 1:1,000 with the HRP-secondary conjugate at 1:100,000. Five-percent nonfat milk with 0.05% Tween*-20 was used for blocking. Additional dry ice and/or freight charges Additional hazardous handling charge *Trademark, see Trademark Index To order or for technical support, contact your local office or distributor. See pages -2, 3. 13

38 Western Blotting Stripping Buffers Restore PLUS Western Blot Stripping Buffer A second formulation especially for high-affinity antibodies that require special treatment. When researchers require a robust but gentle Western blotting stripping buffer, the original Thermo Scientific Restore Western Blot Stripping Buffer has been the buffer of choice (see previous page). However, some antibodies remain difficult to remove from Western blots and require longer incubation times or incubation temperatures greater than 22 C. Thermo Scientific Restore PLUS Western Blot Stripping Buffer was developed to reduce incubation times while keeping incubations at room temperature. High-affinity antibodies can be quickly and effectively stripped from Western blots without removing transferred proteins, thereby allowing multiple reprobes of the target. Original Stripped Reprobed Thermo Scientific Restore PLUS Buffer Supplier A Supplier C Reprobing with different antibodies. HeLa cell lysate was probed for actin and detected with Thermo Scientific Pierce ECL Substrate (Original panel). Blots were then stripped with either Restore PLUS Stripping Buffer or other suppliers stripping buffers (Stripped panel). The blots were then re-blocked and reprobed for cyclophilin B and detected with Pierce ECL Substrate (Reprobed panel). Western Blotting Technical Handbook Actin Actin Actin Cyclophilin B Ready and easy to use No dilution necessary No offensive odors Store at room temperature Compatible with commonly used Western blotting reagents and other materials Use on nitrocellulose and PVDF membranes, stored wet or dry Works with blocking buffer, enzyme conjugate and chemiluminescent substrate of choice Cost effective Save valuable time and samples Strip blots effectively the first time Robust, but gentle Transferred proteins remain viable Strip the same blot up to five times Flexible Strip and re-probe to optimize antibody concentrations Strip and re-probe for new antigen of interest 4428 Restore PLUS Western Blot 30 ml Stripping Buffer Sufficient reagent to strip one to two (8 cm x 10 cm) blots Restore PLUS Western Blot Stripping Buffer Sufficient reagent to strip 25 (8 cm x 10 cm) blots. 500 ml Western Blotting Handbook and Troubleshooting Guide Featuring Thermo Scientific SuperSignal Substrates and Pierce Western Blotting Accessories The Western Blotting Handbook and Troubleshooting Guide is a 75-page booklet that details each step of the Western blotting process with technical information and products for transfer, blocking, washing, antibodies, substrates, film and stripping buffer. You will want to keep this booklet close at hand because it also includes protocols, references and a troubleshooting guide. To download a copy or view a pdf online, visit To request a printed copy, visit the same website or contact your local office or distributor. 14 To order or for technical support, contact your local office or distributor. See pages -2, 3.

39 Western Blotting Colorimetric Substrates Precipitating Colorimetric Substrates for HRP As with the other components in a Western blotting system, there are many substrate choices available. The appropriate substrate choice depends on the enzyme label (AP or HRP), desired sensitivity, and desired form of signal or method of detection. Chromogenic substrates are widely used and offer perhaps the simplest and most cost-effective method of detection. When these substrates come in contact with the appropriate enzyme, they are converted to insoluble, colored products that precipitate onto the membrane and require no special equipment for processing or visualizing. Substrates such as TMB (3,3,5,5 -tetramethylbenzidine), 4-CN (4-chloro-1-naphthol) and DAB (3,3 -diaminobenzidine tetrahydrochloride) are available for use with HRP. Peroxide must be added to a substrate for colorimetric detection with HRP. Because of its extremely short shelf life at the desired concentration, hydrogen peroxide traditionally was added to a buffer, along with the substrate, immediately before use. As a result, these substrates typically have a useful shelf life of only a few hours. Many of our precipitating HRP substrates are supplied with, or come prepared in, Thermo Scientific Pierce Stable Peroxide Substrate Buffer (Product # 3402). The Stable Peroxide Substrate Buffer is a 10X concentrate that offers several advantages over traditional 30% stock solutions of hydrogen peroxide. Pierce Stable Peroxide Buffer Convenient, stable solution for HRP substrates. The Thermo Scientific Pierce Stable Peroxide Substrate Buffer is a 10X concentrate that offers several advantages. It is less corrosive than the traditional 30% stock solution of hydrogen peroxide and, because fewer preparation steps are involved, it provides more consistent results. Although the Stable Peroxide Substrate Buffer is provided as a 10X concentrate, it is also stable at a 1X concentration. Pierce TMB-Blotting Substrate Solution Straight-from-the-bottle convenience. TMB (3,3,5,5 -tetramethylbenzidine), with a molecular weight of 240.4, is most often used as a substrate for HRP in ELISAs. However, in the presence of HRP and peroxide, a water-soluble blue product is generated that can be precipitated onto a membrane. Thermo Scientific Pierce TMB-Blotting is a single-component peroxidase substrate for Western blotting and immunohistochemistry. Precipitating the product results in dark blue bands where the enzyme is located. Pierce TMB-Blotting is well suited to applications that require a high signal-to-noise ratio Pierce Stable Peroxide Buffer (10X) 100 ml Stable, pre-filtered and ready-to-use single component No hydrogen peroxide required Noncarcinogenic and no DMF or DMSO present Large dynamic range, high sensitivity, and minimal fading Best used with Thermo Scientific SuperBlock (TBS) Blocking Buffer (Product # 27517, 37537) Pierce TMB-Blotting Substrate Solution 250 ml Chloronaphthol (CN) Substrates Ready-to-use solution, tablets or powder. 4-CN has a molecular weight of 178. and can be used for chromogenic detection of HRP in blotting and histochemistry. This precipitate is not as sensitive or as stable as TMB and DAB, but the alcohol-soluble precipitate photographs well and has a distinct blue-purple color that can be useful in double-staining applications. Stable, pre-filtered and ready-to-use solution No hydrogen peroxide required Photographs well CN Substrate Solution 250 ml Chloro-1-Naphthol 25 g powder Chloro-1-Naphthol Tablets (30 mg/tablet) 50 tablets Additional dry ice and/or freight charges Additional hazardous handling charge *Trademark, see Trademark Index To order or for technical support, contact your local office or distributor. See pages -2, 3. 15

40 Western Blotting Colorimetric Substrates Pierce CN/DAB Substrate Kit Synergizes the photographic ability of CN with the sensitivity of DAB. The individual benefits of 4-CN and DAB are often combined into a single substrate mixture, CN/DAB Substrate. The Thermo Scientific Pierce CN/DAB Substrate has excellent sensitivity, yielding a dark black precipitate that photographs well. The CN/DAB Substrate works well in Western blotting and dot blotting applications. Reference Young, P.R. (1989). J. Virol. Methods 24, Pierce CN/DAB Substrate Kit Kit Includes: CN/DAB (10X) 25 ml Stable Peroxide Buffer 250 ml DAB Complete kit or formulate your own DAB substrate. DAB (3,3'-diaminobenzidine) has a molecular weight of and yields a brown precipitate in the presence of HRP and peroxide. The brown, insoluble product can be readily chelated with osmium tetroxide, a property that makes Thermo Scientific DAB especially useful for electron microscopy. The brown color does not fade, enabling detected nitrocellulose Western blots to be stored indefinitely. Versatile substrate for blotting and other applications Offered as powder and as 10X solution with peroxide solution Metal Enhanced DAB Substrate Kit Most sensitive DAB substrate formulation available. The color produced by DAB (3,3'-diaminobenzidine) can be intensified by the addition of metals such as nickel, copper, silver and cobalt that form more dense complexes upon reaction in HRP-peroxide substrate reactions. The Thermo Scientific Metal Enhanced DAB Substrate Kit optimizes this intensifying chemistry, producing an exceptionally sensitive colorimetric detection system for Western blot, dot-blot and immunohistochemistry applications. The near-black signal (precipitate) is easy to photograph. Incredible sensitivity 50 times more sensitive than the traditional DAB method and 30 times more sensitive than other metal-intensified versions of DAB! Low background, high intensity get a crisp dark brown-black precipitate and, even with the increased intensity, background is almost nonexistent! Six-hour stability working solution is stable for more than six hours at room temperature! Other DAB substrates must be used immediately Adams, J.C. (1977). Neuroscience 2, Adams, J.C. (1981). J. Histochem. Cytochem. 29, 775. De Jong, A.S., et al. (1985). Histochem. J. 17, Hsu, S. and Soban, E. (1982). J. Histochem. Cytochem. 30, Malmgren, L. and Olsson, Y. (1977). J. Histochem. Cytochem. 25, Scopsi, L. and Larson, L.I. (198). Histochemistry 84, Straus, W. (1982). J. Histochem. Cytochem. 30, DAB 10 g powder DAB Substrate Kit Kit Includes: DAB (10X) 25 ml Stable Peroxide Buffer 250 ml Sensitivity of DAB substrate methods. Substrate DAB Substrate (Method of Graham and Karnovsky) Metal Enhanced DAB Substrate (Method of Adams) Thermo Scientific Metal Enhanced DAB Substrate Kit (Product # 3405) Lower Detectability Limit ng Horseradish peroxidaseconjugated streptavidin ng Horseradish peroxidaseconjugated streptavidin ng Horseradish peroxidaseconjugated streptavidin Adams, J.C. (1977). Neuroscience 2, Adams, J.C. (1981). J. Histochem. Cytochem. 29, 775. Geoghegan, W.D. and Ackerman, G.A. (1977). J. Histochem. Cytochem. 25, Graham, R.C. and Karnovsky, M.J. (19). J. Histochem. Cytochem. 14, Rodbard, D. (1978). Anal. Biochem. 90, Metal Enhanced DAB Substrate Kit Kit Includes: 10X Metal Enhanced DAB 25 ml Stable Peroxide Buffer 250 ml 1 To order or for technical support, contact your local office or distributor. See pages -2, 3.

41 Western Blotting Colorimetric Substrates Precipitating Colorimetric Substrates for Alkaline Phosphatase Although not as sensitive and versatile as chemiluminescent substrates, colorimetric substrates remain useful as simple and cost-effective detection systems for many situations. Colorimetric substrates for alkaline phosphatase (AP) enzyme function by forming a colored product or complex upon cleavage of a phosphate group by the enzyme. NBT, BCIP and the combination thereof result in a dark blue precipitation complex at sites of AP activity. NBT/BCIP reaction scheme. BCIP is hydrolyzed by alkaline phosphatase to form an intermediate that undergoes dimerization to produce an indigo dye. The NBT is reduced to the NBT-formazan by the two reducing equivalents generated by the dimerization. NBT Substrate NBT (nitro-blue tetrazolium chloride), with a molecular weight of 817., is a member of a class of heterocyclic organic compounds known as tetrazolium salts. Upon reduction, the compound yields NBT-formazan, a highly colored, water-insoluble product. The substrate is widely used for immunochemical assays and techniques because the color produced by the formazan is linear and stable over a wide dynamic range. BCIP Substrate BCIP (5-bromo-4-chloro-3 -indolylphosphate p-toluidine salt) has a molecular weight of 433., and hydrolysis by AP results in a blue-purple precipitate. BCIP can be used as a chromogenic substrate for both immunoblotting and immunohistochemical studies. Br CI Br N H BCIP CI O O P OH N OH H O -2H AP Br CI NBT NBT-formazan Br CI N H O N H O N H OH HPO 3 - tautomerism CI Br 5,5'-dibromo-4,4'- dichloro-indigo white NBT Substrate 1 g powder BCIP Substrate 1 g powder NBT/BCIP Substrate Solutions An ideal system for blotting or staining applications with AP is the combination of NBT and BCIP. Together, they yield an intense, blackpurple precipitate that provides much greater sensitivity than either substrate alone. This reaction proceeds at a steady rate, allowing accurate control of its relative sensitivity. NBT/BCIP characteristically produces sharp band resolution with minimal background. Highlights Ready-to-use, single-component solutions Regular formulation ideal for Western blotting Suppressor formulation contains levamisole for inhibition of endogenous enzyme, making it ideal for immunohistochemistry applications Reference Stuyver, L.J., et al. (2003). Antimicrob. Agents Chemother. 47, NBT/BCIP Substrate Solution 250 ml NBT/BCIP Plus Suppressor Substrate Solution 100 ml Additional dry ice and/or freight charges Additional hazardous handling charge *Trademark, see Trademark Index To order or for technical support, contact your local office or distributor. See pages -2, 3. 17

42 ELISA Labware Thermo Scientific ELISA Reagents and Consumables Reagent Reservoirs Allows easy dispensing of reagents by multi-channel pipettors. Reservoirs facilitate repeated multi-channel pipetting for reagent delivery to microplates. Thermo Scientific Reagent Reservoirs are sterile, sturdy, disposable and molded from high-impact polystyrene Reagent Reservoirs Sterile, 50 ml capacity 200/pkg. Sealing Tape for 9-Well Plates These are pre-cut, pressure-sensitive sealing tape sheets for standard size microplates. Seal plates quickly and easily to allow mixing or prevent evaporation during assay incubation steps. Pierce 9-Well Plates Enhance antibody-binding properties in your ELISA applications. Thermo Scientific Pierce 9-Well Microplates are made of specially formulated polystyrene and treated to produce high-capacity antibodybinding characteristics. Each lot is certified to ensure adherence to strict performance criteria Sealing Tape for 9-Well Plates 100/pkg. Characteristics: Antibody Binding: High, 400 ng IgG/cm 2 (1 cm 2 = 100 µl vol.) Binding Interaction: Hydrophobic/Ionic Performance Certification: Well-to-Well, CV 3% Well Shape (bottom): Flat Maximum Well Volume: 30 µl Packaging: 1 plate per sealed tray Pierce 9-Well Plates Corner Notch 100/pkg Pierce White Opaque 9-Well Plates 25/pkg. Pierce 8-Well Strip Plates No more loose-fitting strips! Thermo Scientific Pierce 8-Well Strip Plates are optically clear, polystyrene plates that have a standard 9-well microplate footprint but also allow individual 8-well strips (i.e., plate columns) to be removed from the rigid frame. The format provides convenience and quality-assurance when performing partial-plate assays because unused wells can be removed to prevent confusion and cross-contamination. Characteristics: Antibody Binding: High, 400 ng IgG/cm 2 (1 cm 2 = 100 µl vol.) Binding Interaction: Hydrophobic/Ionic Performance Certification: Well-to-Well, CV 3% Well Shape (bottom): Flat Maximum Well Volume: 30 µl Packaging: 5 plates per sealed tray Pierce 8-Well Strip Plates Corner Notch 100/pkg. Includes one Strip Well Ejector per package Pierce Strip Well Ejector 1/pkg. 18 To order or for technical support, contact your local office or distributor. See pages -2, 3.

43 ELISA Labware Pierce ELIFA System Reduces each step to a five-minute filtration! The Thermo Scientific Pierce ELIFA System is used for performing a rapid, membrane-based form of ELISA called ELIFA (enzyme-linked immunoflow assay). The system consists of a 9-well vacuum manifold for insertion of nitrocellulose or nylon membrane. ELISA-type immuno-assays can be completed in just 25 minutes because each step is reduced to a 5 minute filtration. Greater sensitivity because transfer membranes bind more protein than plastic surfaces 9-well arrangement is compatible with 9-well microplates and multichannel pipettes Most wash steps are eliminated, further reducing assay time Cross-talk between samples is eliminated due to the use of tight seals Results are quantitated easily by simply drawing color solution into a microplate and reading using a standard ELISA reader Use insoluble or precipitating substrates to create dot blot results Sample well Sample application plate Membrane Gaskets Transfer cannula Microplate Collection chamber Thermo Scientific Pierce ELIFA System diagram. Colored product is filtered into microplate wells. Precipitated colored product is deposited on membrane. 1:1 1:2 1:4 1:8 1:1 1:32 1:4 1:1 1:2 1:4 1:8 1:11:321: Checkerboard titration using a precipitating substrate. A checkerboard titration is a single experiment in which the concentration of two components is varied in a way to determine the best signal-to-noise ratio conditions. Liu, Z.-H., et al. (2001). Proc. Natl. Acad. Sci. USA 98, Menalled, L.B., et al. (2002). J. Neurosci. 22, Paffard, S.M., et al. (199). J. Immunol. Methods 192, Pierce ELIFA System Includes: Sample application plate 1 Top silicone gasket 1 Membrane support plate with bottom 1 silicone gasket and 9 cannulas Vacuum chamber with o-ring Thumbscrews One-way valves Valve/tubing adapters 4 Tubing 4 ft. Extra cannulas Replacement Cannulas 100/pkg Replacement Clear 9-Well Silicone Gaskets 4/pkg. Patent, see Patent Index. Related Products Page Biodyne* A Nylon Membranes Biodyne B Nylon Membranes 145 A B C D E F G H 1.1 ml Microtubes and Racked Systems Laboratory essentials that simplify and speed up pipettor applications. The 8 x 12 format simplifies and speeds applications in which a multi-channel pipettor is used Format is also compatible with robotic workstations Thermo Scientific Microtube System can be centrifuged Microtube Racked System 10 racks of 9 individual 1.1 ml tubes per rack 1508 Microtubes 1.1 ml individual Microtubes In-line Microtube Caps 8-cap strips 90 tubes 90 tubes 120 Additional dry ice and/or freight charges Additional hazardous handling charge *Trademark, see Trademark Index To order or for technical support, contact your local office or distributor. See pages -2, 3. 19

44 ELISA Coated Plates Coated Polystyrene Microplates The initial step in many microplatebased assays involves passive adsorption of proteins to the plate wells. During adsorption, problems may arise as a result of improper orientation or denaturation of the protein. Contaminating proteins also may cause problems. Thermo Scientific Pierce Coated Plates are designed to circumvent these problems. We offer many of our coated plates in 9-well and 384-well formats in clear, black or white polystyrene plastic to provide flexibility in detecting assay results. Whether the detection system is a chromogen, a fluorescent label or a chemiluminescent substrate, we are likely to have a suitable plate for your application. We also offer a custom coating service whereby you can specify: Coated 9- or 384-well plates, slides or other coated surfaces A coated plate using a certain type of plate or a specific supplier s plate A specific surface chemistry that is not included in this catalog Capture Antibody Coated Plates For species-specific antibody capture. The Thermo Scientific Pierce Capture Antibody Coated Plates are ideal for binding assays when available antibodies are in low quantities or denature upon direct adsorption to polystyrene plates. Types of Thermo Scientific Pierce Coated Plates. Coated Ligand Protein A or Protein G or Protein A/G Protein L Capture Antibodies Streptavidin or NeutrAvidin Protein Biotin Glutathione Anti-GST Antibody Metal Chelate (Ni 2+, Cu 2+ ) Maleic Anhydride Maleimide Activated Application Prevents antibody denaturation caused by direct binding Unlike Protein A or G, these bind only to target species Antibody-binding capacity is higher than direct adsorption Pre-blocked to reduce nonspecific binding Less expensive than Protein A or G plates Gartner, W., et al. (2001). Cereb Cortex. 11, Wagner, L., et al. (2000). J. Biol. Chem. 275, For binding antibodies via their Fc regions For binding Fab antibody fragments and single-chain variable fragments (ScFvs) through the kappa light chain For binding antibodies, as an alternative to Protein A, G or L For binding biotinylated proteins, peptides or nucleic acids For binding avidin, streptavidin or Thermo Scientific NeutrAvidin Biotin-Binding Proteins For binding recombinantly expressed glutathione S-transferase (GST) tagged proteins For binding recombinantly expressed glutathione S-transferase (GST) tagged proteins For binding recombinantly expressed histidinetagged proteins For covalent binding of both large and small amine-containing molecules For covalent binding of sulfhydryl-containing molecules Product # Coating Plate Type Blocking Buffer Binding Capacity Goat Anti-Mouse IgG Antibody, 100 µl Clear, 9-Well SuperBlock BB, 200 µl ~7 pmol mouse IgG/well 5 plates Goat Anti-Mouse IgG Antibody, 100 µl White, 9-Well SuperBlock BB, 200 µl ~7 pmol mouse IgG/well 5 plates Goat Anti-Mouse IgG Antibody, 100 µl Black, 9-Well SuperBlock BB, 200 µl ~7 pmol mouse IgG/well 5 plates Goat Anti-Rabbit IgG Antibody, 100 µl Clear, 9-Well SuperBlock BB, 200 µl ~2 pmol rabbit IgG/well 5 plates 1513 Goat Anti-Rabbit IgG Antibody, 100 µl White, 9-Well SuperBlock BB, 200 µl ~2 pmol rabbit IgG/well 5 plates Goat Anti-Rabbit IgG Antibody, 100 µl Black, 9-Well SuperBlock BB, 200 µl ~2 pmol rabbit IgG/well 5 plates Approximate values; plates tested for specific signal:noise and C.V. BB = Blocking Buffer Antibody Handbook ANTIBODIES Antikörper Anticuerpos Anticorpi Anticorps Anticorpos Thermo Scientific Pierce Antibody Handbook The antibodies presented in this handbook are just a small portion of our total offering. Visit to select from more than 30,000 antibodies and antibodyrelated literature. 170 To order or for technical support, contact your local office or distributor. See pages -2, 3.

45 ELISA Coated Plates Protein A and Protein G Coated Plates Bind antibodies through their Fc portion, correctly orienting them for maximum antigen capture. Thermo Scientific Pierce Protein A and Protein G Coated Plates retain antibody activity, which can be lost when antibodies are immobilized by passive adsorption. Orient antibodies for maximum antigen-binding capacity Immobilize antibodies without prior purification Ensure consistent coating (< 5% well-to-well variation) Reduce nonspecific binding because plates are pre-blocked Perform plated-based immunoprecipitations Binds up to 2.5 µg antibody/well (100 µl coating volume) For antibody-binding specificities of Protein A and Protein G, see Catalog Section 11, page 389 Protein A Asthagiri, A.R., et al. (1999). J. Biol. Chem. 274, Lawrenson, I.D., et al. (2002). J. Cell Sci. 115, Protein G Lai, Z., et al. (2002). Proc. Natl. Acad. Sci. USA 99, Rauch, J., et al. (2003). J. Biol. Chem. 278, Pierce Protein L Coated Plates Great for binding ScFv and Fab fragments. Thermo Scientific Pierce Protein L Coated Plates are pre-blocked to reduce nonspecific binding. Binds to all classes of Ig (IgG, IgM, IgA, IgE and IgD) Binds to the V L region of kappa light chains (human I, III, IV and Mouse I) without interfering with antigen-binding sites Binds ScFvs Does not bind bovine, goat or sheep Igs Binds weakly to rabbit Igs Absorbance (450 nm) Protein A Protein G Passively Bound Antibody Antibody Added per Well (ng) Properly oriented capture antibodies retain higher activity in ELISA. Product # Coating Plate Type Blocking Buffer Binding Capacity Protein A, 100 µl Clear, 9-Well SuperBlock BB, 200 µl ~4 pmol rabbit IgG/well 5 plates Protein A, 100 µl Clear, 8-Well Strip SuperBlock BB, 200 µl ~4 pmol rabbit IgG/well 5 plates Protein A, 100 µl White, 9-Well SuperBlock BB, 200 µl ~4 pmol rabbit IgG/well 5 plates Protein A, 100 µl Black, 9-Well SuperBlock BB, 200 µl ~4 pmol rabbit IgG/well 5 plates Protein G, 100 µl Clear, 9-Well SuperBlock BB, 200 µl ~2 pmol rabbit IgG/well 5 plates Protein G, 100 µl Clear, 8-Well Strip SuperBlock BB, 200 µl ~2 pmol rabbit IgG/well 5 plates 1515 Protein G, 100 µl White, 9-Well SuperBlock BB, 200 µl ~2 pmol rabbit IgG/well 5 plates Protein G, 100 µl Black, 9-Well SuperBlock BB, 200 µl ~2 pmol rabbit IgG/well 5 plates Protein A/G, 100 µl Clear, 8-Well Strip SuperBlock BB, 200 µl ~5 pmol rabbit IgG/well 5 plates Approximate values; plates tested for specific signal:noise and C.V. BB = Blocking Buffer Åkerström, B. and Björck, L. (1989). J. Biol. Chem. 24, Björck, L. (1988). J. Immunol. 140, Kastern, W., et al. (1992). J. Biol. Chem. 27, Nilson, B.H.K., et al. (1993). J. Immunol. Methods 14, Rhee, J., et al. (2001). J. Biol. Chem. 27, Product # Coating Plate Type Blocking Buffer Binding Capacity Protein L, 100 µl Clear, 9-Well SuperBlock BB, 200 µl ~ 3 pmol mouse IgG/well 5 plates Approximate values; plates tested for specific signal:noise and C.V. BB = Blocking Buffer Additional dry ice and/or freight charges Additional hazardous handling charge *Trademark, see Trademark Index To order or for technical support, contact your local office or distributor. See pages -2,

46 ELISA Coated Plates NeutrAvidin Coated Polystyrene Plates The high affinity of avidin for biotin, without the nonspecific binding problems. Comparison of biotin-binding proteins. Protein Isoelectric Point Contains Carbohydrate Nonspecific Binding Avidin Yes High Streptavidin 5 No Low NeutrAvidin Protein.3 No Ultralow Brett, P.J., et al. (2002). J. Biol. Chem. 277, Denlinger, L.C., (2001). J. Immunol. 17, Guixiang Dai, G., et al. (2002). J. Biol. Chem. 277, Patil, S., et al. (1999). J. Biol. Chem. 274, Singh, Y., et al. (1999). Infect. Immun. 7, Easy and gentle immobilization of biotin-containing conjugates Lowest nonspecific binding properties of all biotin-binding proteins Thermo Scientific NeutrAvidin Biotin-Binding Protein has no carbohydrate and an isoelectric point of.3 No denaturing of the protein component of a conjugate upon binding to the plate Ideal for binding small hydrophilic molecules (e.g., peptides) that typically exhibit poor binding directly to polystyrene Pre-blocked with your choice of Thermo Scientific Blocker BSA or SuperBlock Blocking Buffer Available in 9- and 384-well formats Net Absorbance (450 nm) Units src kinase Purified p0 c-src Activity Detection with TK Peptide 2. Biotinylated tyrosine kinase peptide 2 was added to NeutrAvidin Coated Plates and incubated for 30 minutes. Wells were washed; samples containing p0c-src tyrosine kinase were added to phosphorylate the tyrosine residue on the peptide. Anti-phosphotyrosine monoclonal antibody conjugated to horseradish peroxidase was added. Tyrosine kinase activity was detected by Thermo Scientific Turbo-TMB Substrate. Kinase activity was quantitated by comparison with a standard curve generated using the phosphorylated form of the same peptide substrate. Product # Coating Plate Type Blocking Buffer Binding Capacity NeutrAvidin Protein, 100 µl Clear, 9-Well SuperBlock BB, 200 µl ~15 pmol biotin/well 5 plates NeutrAvidin Protein, 100 µl Clear, 8-Well Strip SuperBlock BB, 200 µl ~15 pmol biotin/well 5 plates NeutrAvidin Protein, 50 µl Clear, 384-Well SuperBlock BB, 100 µl ~10 pmol biotin/well 5 plates 1511 NeutrAvidin Protein, 100 µl White, 9-Well SuperBlock BB, 200 µl ~15 pmol biotin/well 5 plates NeutrAvidin Protein, 50 µl White, 384-Well SuperBlock BB, 100 µl ~10 pmol biotin/well 5 plates NeutrAvidin Protein, 100 µl Black, 9-Well SuperBlock BB, 200 µl ~15 pmol biotin/well 5 plates NeutrAvidin Protein, 50 µl Black, 384-Well SuperBlock BB, 100 µl ~10 pmol biotin/well 5 plates NeutrAvidin Protein, 200 µl Clear, 9-Well Blocker BSA, 300 µl ~15 pmol biotin/well 5 plates NeutrAvidin Protein, 200 µl Clear, 8-Well Strip Blocker BSA, 300 µl ~15 pmol biotin/well 5 plates 1521 NeutrAvidin Protein, 200 µl White, 9-Well Blocker BSA, 300 µl ~15 pmol biotin/well 5 plates NeutrAvidin Protein, 200 µl Black, 9-Well Blocker BSA, 300 µl ~15 pmol biotin/well 5 plates Pierce Biotin Binding Plate Sample Pack, one each of # 15120, 15121, 15127, plates Approximate values; plates tested for specific signal: noise and C.V. BB = Blocking Buffer 172 To order or for technical support, contact your local office or distributor. See pages -2, 3.

47 ELISA Coated Plates NeutrAvidin High Binding Capacity (HBC) Coated Plates Unique technology for improved assay precision. We offer researchers a wide variety of avidin-biotin products, including our exclusive Thermo Scientific NeutrAvidin Coated Plates available in a high binding capacity (HBC) format. NeutrAvidin* Protein is a deglycosylated form of avidin with a near-neutral pi that results in less nonspecific binding than that of streptavidin or avidin. Our patent-pending plate-coating technology offers a NeutrAvidin HBC Plate with a wider detection limit than our regular binding capacity plates. The standard curve exhibits greater linearity for detecting small biotinylated molecules such as peptides (see Figure) and oligonucleotides, resulting in greater assay precision. Try NeutrAvidin HBC Coated Plates for binding small biotinylated ligands and see the difference. Unique plate-coating technology results in high loading of NeutrAvidin Protein per well Improved sensitivity less nonspecific binding for improved signal-tonoise ratios Broader dynamic range extends the quantitative range so there s no need for dilutions Save time pre-blocked plates to reduce the number of assay steps Flexible assay formats coated plates offered in 9- and 384-well formats and in different colors HBC RBC CHC Biotinylated Phosphopeptide (pm/well) Comparison of Thermo Scientific NeutrAvidin High Binding Capacity (HBC) Coated Plate, NeutrAvidin Regular Binding Capacity (RBC) Coated Plates and another supplier s Streptavidin Coated High Binding Capacity Plates (CHC). Plates were incubated with various dilutions of biotinylated, phosphorylated peptide. After washing, the plates were incubated with mouse anti-phosphotyrosine antibody (1:1,000) and then detected using an anti-mouse-fitc conjugate (1:). Reference Blouin, C.C., et al. (2004). Blood 103, Product # Coating Plate Type Blocking Buffer Binding Capacity NeutrAvidin Protein, 100 µl Clear, 9-Well SuperBlock BB, 200 µl ~0 pmol biotin/well 5 plates NeutrAvidin Protein, 100 µl Clear, 8-Well Strip SuperBlock BB, 200 µl ~0 pmol biotin/well 5 plates NeutrAvidin Protein, 50 µl Clear, 384-Well SuperBlock BB, 100 µl ~35 pmol biotin/well 5 plates NeutrAvidin Protein, 100 µl White, 9-Well SuperBlock BB, 200 µl ~0 pmol biotin/well 5 plates NeutrAvidin Protein, 50 µl White, 384-Well SuperBlock BB, 100 µl ~35 pmol biotin/well 5 plates NeutrAvidin Protein, 100 µl Black, 9-Well SuperBlock BB, 200 µl ~0 pmol biotin/well 5 plates NeutrAvidin Protein, 50 µl Black, 384-Well SuperBlock BB, 100 µl ~35 pmol biotin/well 5 plates Approximate values; plates tested for specific signal:noise and C.V. BB = Blocking Buffer Signal-to-noise Ratio Additional dry ice and/or freight charges Additional hazardous handling charge *Trademark, see Trademark Index To order or for technical support, contact your local office or distributor. See pages -2,

48 ELISA Coated Plates Streptavidin Coated Plates The specific binding affinity of streptavidin for biotin in a microplate. The Thermo Scientific Pierce Streptavidin Coated Plates are made of polystyrene and are ideal for binding assays using biotinylated molecules. Streptavidin has no carbohydrate groups and an isoelectric point of 5-, resulting in low nonspecific interactions. Easy and gentle immobilization of biotin-containing conjugates Low nonspecific binding No denaturing of the protein component of a conjugate upon binding Ideal for binding small biotinylated hydrophilic molecules (e.g., peptides) that typically exhibit poor binding to polystyrene Pre-blocked with your choice of Thermo Scientific Blocker BSA or SuperBlock Blocking Buffer Available in clear, white and black plates in 8-well strip, 9-well and 384-well formats Estrada, G., et al. (199). Mol. Cell Probes 10, Grobler, J.A., et al. (2002). Proc. Natl. Acad. Sci. USA 99, 1-. Hong, P. W.-P., et al. (2002). J. Virol. 7, Stühlinger, M.C., et al. (2001). Circulation 104, Su, S.V., et al. (2004). J. Biol. Chem. 279(18), Product # Coating Plate Type Blocking Buffer Binding Capacity Streptavidin, 100 µl Clear, 9-Well SuperBlock BB, 200 µl ~5 pmol biotin/well 5 plates 1512 Streptavidin, 100 µl Clear, 9-Well SuperBlock BB, 200 µl ~5 pmol biotin/well 5 x 5 plates Streptavidin, 100 µl Clear, 8-Well Strip SuperBlock BB, 200 µl ~5 pmol biotin/well 5 plates Streptavidin, 100 µl Clear, 8-Well Strip SuperBlock BB, 200 µl ~5 pmol biotin/well 5 x 5 plates Streptavidin, 50 µl Clear, 384-Well SuperBlock BB, 100 µl ~4 pmol biotin/well 5 plates Streptavidin, 100 µl White, 9-Well SuperBlock BB, 200 µl ~5 pmol biotin/well 5 plates Streptavidin, 100 µl Black, 9-Well SuperBlock BB, 200 µl ~5 pmol biotin/well 5 plates Streptavidin, 50 µl Black, 384-Well SuperBlock BB, 100 µl ~4 pmol biotin/well 5 plates Streptavidin, 200 µl Clear, 9-Well Blocker BSA, 300 µl ~10 pmol biotin/well 5 plates Streptavidin, 200 µl Clear, 8-Well Strip Blocker BSA, 300 µl ~10 pmol biotin/well 5 plates Streptavidin, 200 µl White, 9-Well Blocker BSA, 300 µl ~10 pmol biotin/well 5 plates Streptavidin, 200 µl Black, 9-Well Blocker BSA, 300 µl ~10 pmol biotin/well 5 plates Pierce Biotin Binding Plate Sample Pack, one each of # 15120, 15121, 15127, plates Approximate values; plates tested for specific signal:noise and C.V. BB = Blocking Buffer Do you need Coated 9- or 384-well plates, slides or other coated surfaces? A coated plate using a certain type of plate or a specific supplier s plate? A specific surface chemistry that is not included in this catalog? We can help. To learn about our Plate-Coating Service, please call or us and ask for Bulk & Custom Sales. All Thermo Scientific Pierce Coated 9- and 384-well plates are available in bulk quantities with discounted pricing. Contact us for bulk pricing information and packaging options. 174 To order or for technical support, contact your local office or distributor. See pages -2, 3.

49 ELISA Coated Plates Streptavidin High Binding Capacity (HBC) Coated Plates Take advantage of our technology that provides a broader dynamic range. Thermo Scientific Pierce Streptavidin High Binding Capacity (HBC) Coated Plates are designed for binding biotinylated oligonucleotides and peptides with higher binding efficiency than other commercially available plates. Our proprietary coating technology (patent pending) has created a streptavidin-coated plate with four- to five-times the binding capacity of other suppliers plates. Using a Pierce Streptavidin HBC Plate can result in an assay with a broader dynamic range and better linearity, leading to improved assay precision. Try our Streptavidin HBC Coated Plate and see what has been going undetected in your research. Broader dynamic range extends the quantitative range so there s no need for dilutions Better sensitivity increased binding capacity allows direct detection of small ligands not observed with regular binding capacity plates Superior assay precision standard curve demonstrates greater linearity Save time pre-blocked to reduce number of assay steps Flexible assay formats offered in 9- and 384-well formats and in different colors Biotin Coated Plates A simple format for testing the efficiency of avidin, streptavidin or Thermo Scientific NeutrAvidin Protein conjugations. Thermo Scientific Pierce Biotin Coated Plates can be used in any immunoassay with Thermo Scientific NeutrAvidin Biotin-Binding Protein, streptavidin, avidin or other biotin-binding proteins. Biotin group accessible for binding avidin, streptavidin or NeutrAvidin* Biotin-Binding Protein Pre-blocked to reduce nonspecific binding Strip well plate format for the ultimate in convenience Signal-to-noise Ratio Thermo Scientific Pierce Streptavidin HBC Plates Competing high capacity plates Fluoresceinated Oligonucleotide (pm/well) Comparison of Thermo Scientific Pierce Streptavidin High Binding Capacity (HBC) Coated Plate with competing high binding capacity plate. Plates were incubated with a biotinylated oligonucleotide, washed and probed with a complementary oligonucleotide labeled with fluorescein at various dilutions. Reference Wilson, D.S., et al. (2001). Proc. Natl. Acad. Sci. USA 98, Product # Coating Plate Type Blocking Buffer Binding Capacity Streptavidin, 100 µl Clear, 9-Well SuperBlock BB, 200 µl ~125 pmol biotin/well 5 plates Streptavidin, 100 µl Clear, 8-Well Strip SuperBlock BB, 200 µl ~125 pmol biotin/well 5 plates Streptavidin, 50 µl Clear, 384-Well SuperBlock BB, 100 µl ~0 pmol biotin/well 5 plates Streptavidin, 100 µl White, 9-Well SuperBlock BB, 200 µl ~125 pmol biotin/well 5 plates Streptavidin, 50 µl White, 384-Well SuperBlock BB, 100 µl ~0 pmol biotin/well 5 plates Streptavidin, 100 µl Black, 9-Well SuperBlock BB, 200 µl ~125 pmol biotin/well 5 plates 1550 Streptavidin, 50 µl Black, 384-Well SuperBlock BB, 100 µl ~0 pmol biotin/well 5 plates Approximate values; plates tested for specific signal:noise and C.V. BB = Blocking Buffer Pierce Biotin Coated Clear 8-Well Strip Plates Coat volume: 200 µl Blocking volume: 300 µl 5 plates Additional dry ice and/or freight charges Additional hazardous handling charge *Trademark, see Trademark Index To order or for technical support, contact your local office or distributor. See pages -2,

50 ELISA Coated Plates Nickel Coated Plates Bind recombinantly expressed fusion proteins containing a histidine tag for easy ELISA analysis. Thermo Scientific Pierce Nickel Coated Plates provide a simple format for protein:protein interaction studies. Detergents used to lyse cells do not inhibit binding to Ni 2+ coated plates as they do with plain polystyrene The detection limit is 1 ng of histidine fusion protein Better binding for more sensitive assays compared to other commercially available nickel-activated plates Can be custom-made with other metals. Contact our Bulk & Custom Sales Personnel or your local distributor Desai, S., et al. (1997). Previews 1(4), Pan, W., et al. (2003). J. Biol. Chem. 278, Tu, Y., et al. (1999). Mol. Cell. Biol. 19, Copper Coated, High Binding Capacity Plates Boost assay performance by binding more histidine-tagged protein. Thermo Scientific Pierce Copper Coated High Binding Capacity (HBC) Plates use an exclusive coating process to increase the amount of histidine-tagged protein that will bind to the plate surface. This is ideal for high throughput screening applications that need improved sensitivity and greater dynamic range. Quantitate previously undetectable histidine-tagged proteins Wider dynamic range with four-fold greater capacity than regular nickel chelate-coated plates Copper chelate provides greater binding capacity than nickel Absorbance (450 nm) Thermo Scientific Supplier Q Nanograms ATF per well Binding comparison of histidine-tagged ATF fusion protein as detected by an HRP-conjugated anti-atf antibody and TMB substrate. Related Products Page # 8998 HisPur Cobalt Spin Columns HisPur Purification Kit 32 Product # Coating Plate Type Blocking Buffer Binding Capacity Nickel Chelate, 200 µl Clear, 9-Well BSA, 200 µl ~9 pmol His-tagged protein/well 5 plates Nickel Chelate, 200 µl Clear, 8-Well Strip BSA, 200 µl ~9 pmol His-tagged protein/well 5 plates Nickel Chelate, 200 µl White, 9-Well BSA, 200 µl ~9 pmol His-tagged protein/well 5 plates Nickel Chelate, 200 µl Black, 9-Well BSA, 200 µl ~9 pmol His-tagged protein/well 5 plates Approximate values; plates tested for specific signal:noise and C.V. Relative Fluorescence Units (RFU) 0,000 50,000 40,000 30,000 20,000 10, pmol Copper Coated HBC Nickel Coated 8.9 pmol Units of HT Fusion Protein Applied Binding comparison of a histidine-tagged fluorescent fusion protein to standard Thermo Scientific Pierce Nickel Coated and Pierce Copper Coated High Binding Capacity (HBC) Plates. Pierce Copper Coated HBC Plates exhibit a four-fold greater capacity for binding purified histidine-tagged protein when assayed using a 100 µl volume. Incubation time was two hours for the binding of histidine-tagged fusion protein. Reference Montes de Oca, R., et al. (2005). J. of Biol. Chem. 280 #51, Product # Coating Plate Type Blocking Buffer Binding Capacity Copper Chelate, 200 µl Clear, 9-Well BSA, 200 µl ~35 pmol His-tagged protein/well 5 plates 1514 Copper Chelate, 200 µl Clear, 8-Well Strip BSA, 200 µl ~35 pmol His-tagged protein/well 5 plates Copper Chelate, 200 µl White, 9-Well BSA, 200 µl ~35 pmol His-tagged protein/well 5 plates Copper Chelate, 200 µl Black, 9-Well BSA, 200 µl ~35 pmol His-tagged protein/well 5 plates Approximate values; plates tested for specific signal:noise and C.V. 17 To order or for technical support, contact your local office or distributor. See pages -2, 3.

51 ELISA Coated Plates Glutathione Coated Plates Bind fusion proteins containing GST. Thermo Scientific Pierce Glutathione Coated Plates have a lower detection limit of 1 ng of GST fusion protein. Easy quantitation of antibodies against GST-fused proteins Simple format for protein:protein interaction studies Detergents used to lyse cells do not inhibit binding Pre-blocked to reduce nonspecific binding McKevitt, M., et al. (2003). Genome Res. 13, Pullen, S.S., et al. (1999). J. Biol. Chem. 274, Yarwood, S.J., et al. (1999). J. Biol. Chem. 274, Product # Coating Plate Type Blocking Buffer Binding Capacity Glutathione, 100 µl Clear, 8-Well Strip SuperBlock BB, 200 µl ~10 ng GST protein/well 5 plates Glutathione, 100 µl White, 9-Well SuperBlock BB, 200 µl ~10 ng GST protein/well 5 plates Glutathione, 100 µl Black, 9-Well SuperBlock BB, 200 µl ~10 ng GST protein/well 5 plates Approximate values; plates tested for specific signal:noise and C.V. BB = Blocking Buffer Anti-GST Coated Plates A unique alternative to glutathione-coated plates for binding GST fusion proteins. Thermo Scientific Pierce Anti-GST Coated Plates bind native or denatured forms of GST. They are an alternative to glutathione-coated plates. Pre-blocked and ready to use Compatible with Thermo Scientific B-PER, M-PER and Y-PER Protein Extraction Reagents Absorbance (450 nm) ,000 Purified GST Concentration (ng/ml) Binding of GST to Thermo Scientific Pierce Anti-GST Coated Plates. Different amounts of purified GST were added to the plate. After washing, bound GST was detected using a rabbit anti-gst antibody (1 mg/ml) followed by donkey anti-rabbit HRP conjugate. The signal was developed with Thermo Scientific TMB Substrate (Product # 34022). Product # Coating Plate Type Blocking Buffer Binding Capacity Goat Anti-GST Antibody, 100 µl Clear, 8-Well Strip SuperBlock BB, 200 µl ~10 ng GST protein/well 5 plates Approximate values; plates tested for specific signal:noise and C.V. BB = Blocking Buffer Additional dry ice and/or freight charges Additional hazardous handling charge *Trademark, see Trademark Index To order or for technical support, contact your local office or distributor. See pages -2,

52 . ELISA Reactive Plates Amine-binding Plates (Maleic Anhydride Activated) Proteins and other primary amine-containing compounds covalently attach to the microplate. Thermo Scientific Pierce Amine-binding Plates are great for immobilization of compounds that do not readily bind to plain polystyrene plates. Spontaneously react with primary amines Maleic anhydride retains its integrity and coupling availability for months Aufderheide, A.C., et al. (2004). Proc. Natl. Acad. Sci. USA 101, Gregory,K. and Mello, C.M. (2005). Appl. Envir. Microbiol. 71, Kojima, K. et al. (2004). J. Am. Soc. Nephrol. 15, Leu, S.-J., et al. (2003). J. Biol. Chem. 278, Li, Y., et al. (2003). J. Biol. Chem. 278, H 2 N Peptide H 2 N Peptide O O O Maleic Anhydride Activated Plate Reaction scheme for coupling amine-containing molecules to Thermo Scientific Maleic Anhydride Plates. O Sulfhydryl-binding Plates (Maleimide Activated) O O - O O A convenient alternative to amine-reactive chemistries for attaching SH containing compounds. Maleimide groups specifically and covalently conjugate sulfhydryl groups at neutral ph, creating a stable thioether bond. Thermo Scientific Pierce Sulfhydryl-binding Plates are ideal for binding molecules that are difficult to coat onto polystyrene plates. Easy (spontaneous) immobilization of peptides derivatized with a terminal cysteine and proteins with free sulfhydryl Pre-blocked to reduce nonspecific binding Horswill, A.R., et al. (2004). Proc. Natl. Acad. Sci. USA 101, Nisitani, S., et al. (1999). Proc. Natl. Acad. Sci. USA 9, Ostrowski, M., et al. (2002). J. Virol. 7, Reinhard, M., et al. (1999). J. Biol. Chem. 274, ph 8-9 ph 3-4 O H N Peptide Immobilized Peptide (ready for use in assay at ph > 7) O HO O Reaction scheme for coupling sulfhydryl-containing molecules to Thermo Scientific Sulfhydryl-binding Plates. OH Hydrolyzed Product (peptide released) Product # Coating Plate Type Binding Capacity Maleic Anhydride, 200 µl Clear, 9-Well ~125 pmol biotin-pentylamine/well 5 plates Maleic Anhydride, 200 µl Clear, 9-Well ~125 pmol biotin-pentylamine/well 5 x 5 plates Maleic Anhydride, 200 µl Clear, 8-Well Strip ~125 pmol biotin-pentylamine/well 5 plates Maleic Anhydride, 200 µl Clear, 8-Well Strip ~125 pmol biotin-pentylamine/well 5 x 5 plates Maleic Anhydride, 200 µl White, 9-Well ~125 pmol biotin-pentylamine/well 5 plates Approximate values; plates tested for specific signal:noise and C.V. O N O O N Maleimide Activated Plate O HS Peptide ph O N S O Peptide Immobilized Peptide Product # Coating Plate Type Blocking Buffer Binding Capacity Maleimide, 200 µl Clear, 8-Well Strip BSA, 200 µl ~ pmol peptide-sh/well 5 plates Maleimide, 200 µl White, 9-Well BSA, 200 µl ~ pmol peptide-sh/well 5 plates Maleimide, 200 µl Black, 9-Well BSA, 200 µl ~ pmol peptide-sh/well 5 plates Approximate values; plates tested for specific signal:noise and C.V. 178 To order or for technical support, contact your local office or distributor. See pages -2, 3.

53 ELISA Blocking Buffers Blocking Buffer Reagents for ELISA Choose the ideal blocker to generate high signal:noise ratios! We offer a variety of blocking buffer formulations to help improve ELISA results. After coating a microplate with a target antigen or capture antibody, a blocking buffer is then incubated in each well to cover nonspecific binding sites. This helps reduce the amount of background or noise in the final assay. The blocking buffer is also used to dilute the assay reagents, such as the primary antibody or antibody-enzyme conjugates, to further reduce any problems associated with nonspecific binding. The result is a higher signal:noise ratio that can improve assay sensitivity and dynamic range. Improve your assay processing time with Thermo Scientific StartingBlock or SuperBlock Blocking Buffers. Blocking can be accomplished in as little as 10 minutes with SuperBlock* Blocking Buffer or with no waiting at all using StartingBlock* Blocking Buffer. Blocked plates can be dried and stored for -12 months at 4 C before performing the assay. Both the StartingBlock and SuperBlock Buffers provide long-term stability to the antibody or other protein coated on the plate. The defined, non-serum protein formulation of each of these blocking buffers ensures less antibody cross-reactivity than milk, BSA, casein and other blocking buffers. Remember that each step of an ELISA must be optimized to render a robust assay system. Selecting the appropriate blocking buffer can make all the difference in how well your ELISA performs over time. Guide to Thermo Scientific Pierce Blocking Buffers. PBS = phosphate buffered saline; TBS = Tris buffered saline; T20 = Tween*-20 Detergent Product # Blocking Buffer StartingBlock (PBS) Blocking Buffer 1 L StartingBlock (TBS) Blocking Buffer 1 L StartingBlock T20 (PBS) Blocking Buffer 1 L StartingBlock T20 (TBS) Blocking Buffer 1 L Protein-Free (TBS) Blocking Buffer 1 L Protein-Free T20 (TBS) Blocking Buffer 1 L Protein-Free (PBS) Blocking Buffer 1 L Protein-Free T20 (PBS) Blocking Buffer 1 L SuperBlock (PBS) Blocking Buffer 1 L SuperBlock (TBS) Blocking Buffer 1 L 3751 SuperBlock T20 (PBS) Blocking Buffer 1 L 3753 SuperBlock T20 (TBS) Blocking Buffer 1 L SuperBlock (TBS) Dry Blend Blocking Buffer 5 pouches (makes 1 L) SEA BLOCK Blocking Buffer 500 ml Blocker BSA in TBS 125 ml (10X) Blocker BSA in PBS 200 ml (10X) Blocker Casein in PBS 1 L Blocker Casein in TBS 1 L Blocker BLOTTO in TBS 1 L For a more detailed description and listing of these blocking buffer products, see pages Blocking Buffer Description ELISA Western Blot and Dot Blot Immunohistochemistry StartingBlock Blocking Buffers Single purified protein; fast blocking; broad applicability; excellent for stripping and reprobing Western blotting applications; available in PBS and TBS with and without T20 SuperBlock Blocking Buffers Single purified glycoprotein; fast blocking; broad applicability; stabilizes plate-coated antibodies for drying; available in PBS and TBS with and without T20 DNA/RNA Hybridizations Blocker BSA Blocking Buffers Purified bovine serum albumin in PBS or TBS Blocker Casein Blocking Buffers Purified casein in PBS or TBS Blocker BLOTTO Blocking Buffer Non-fat dry milk proteins in TBS SEA BLOCK Blocking Buffer Steelhead salmon serum Protein-Free Blocking Buffers Proprietary non-protein blocking compound; available in PBS and TBS with and without T20 Additional dry ice and/or freight charges Additional hazardous handling charge *Trademark, see Trademark Index To order or for technical support, contact your local office or distributor. See pages -2,

54 ELISA Substrates Choosing an Enzyme and Detection Method for Plate-Based Assays A detection step using an enzyme substrate is the final stage in all enzyme immunoassay systems. When substrate is added, a colorimetric, luminescent or fluorescent signal is produced. The signal intensity should correlate to the concentration of the primary antibody and the respective antigen. Choosing an enzyme for ELISA. Enzyme Horseradish Peroxidase Alkaline Phosphatase β-galactosidase Abbreviation HRP AP or Alk. Phos. β-gal Price Relatively inexpensive Relatively expensive Relatively inexpensive Stability Excellent Poor Good Choice of Substrates for ELISA Many Few Several Size (MW) 40, , ,000 ph Optimum Disadvantages Advantages Enzyme-labeled reagents are detected using chromogenic, chemiluminescent or chemifluorescent substrates. Chromogenic substrates are generally the least expensive, while luminescent or fluorescent substrates are often more sensitive. When performing ELISA assays, a soluble substrate is used and converted to a soluble end product. ELISAs are performed on polystyrene plates, and the enzyme levels are Choosing a detection signal type. Properties in ELISA Sensitive to azide; inhibited by cyanide and sulfide Low molecular weight; good for immunocytochemical staining Large, very sensitive to freeze/thaw; need to avoid phosphate-containing buffers Can withstand high temperatures; good for hybridization Very bulky molecule; more easily inactivated than other enzymes Contains abundant sulfhydryl molecules for conjugating with a heterobifunctional molecule Mammalian β-galactosidases have ph optimum in the range of 5.5- (can avoid interference from endogenous β-galactosidase by keeping at ph 7-7.5) determined by monitoring signal development with a spectrophotometer (a luminometer for luminescence or a fluorometer for fluorescence). When performing immunoblotting or immunohistochemical studies, a soluble substrate is used and converted to an insoluble end product. When performing immunoblotting, the antigens are located on nitrocellulose or PVDF membranes that require a precipitating substrate. Colorimetric Substrates Fluorescent Substrates Chemiluminescent Substrates Medium/low sensitivity Generally less expensive Many substrates available Slow signal generation Enzyme catalyzed quickly Small linear range/poor low-end linearity Flexible (stopped, nonstopped and kinetic assays) High sensitivity Generally more expensive Few substrates available Rapid signal generation Enzyme activity maintained Large linear range/enhanced low-end linearity Flexible (stopped, nonstopped and kinetic assays) High sensitivity Generally more expensive Few substrates available Rapid signal generation Enzyme catalyzed quickly Large linear range/enhanced low-end linearity Nonflexible Detection Equipment Spectrophotometer provides quantifiable results Fluorometer provides quantifiable results Luminometer provides quantifiable results Recommended Clear Opaque (black) Opaque (white) Plate Type Enzyme Systems HRP HRP HRP Available AP β-gal 180 To order or for technical support, contact your local office or distributor. See pages -2, 3.

55 ELISA Chemiluminescent Substrates Chemiluminescent HRP Substrates for ELISA Researchers looking for greater sensitivity in their ELISAs or any other solution-based assay are now able to use chemiluminescent substrates for enzyme detection and quantification. These ELISAs can be performed in either a test tube or a microplate and are quantified by measuring relative light units (RLU) in a luminometer. Thermo Scientific SuperSignal ELISA Pico Chemiluminescent Substrate was developed for researchers who need high sensitivity at an economical price. SuperSignal ELISA Femto Maximum Sensitivity Chemiluminescent Substrate uses an improved enhancer system that meets the needs of high-throughput screening and diagnostic customers. Both have their own unique features as listed in the following table. Guide to Thermo Scientific SuperSignal ELISA Substrates. Substrate Detection Limits Kinetics SuperSignal ELISA Femto Quantitative to the femtogram level Immediate light generation Maximum Sensitivity Substrate 5- to 30-minute stability, depending on HRP concentration SuperSignal ELISA Pico Chemiluminescent Substrate SuperSignal ELISA Pico Chemiluminescent Substrate Get sensitivity and a large dynamic range. Quantitative to the picogram level Thermo Scientific SuperSignal ELISA Pico Chemiluminescent Substrate is optimized to generate an intense light signal and provide exception performance in luminometer-based assays. Immediate light generation intense signal is produced immediately at room temperature or at 37 C High signal:noise ratio minimal background Low picogram sensitivity detect proteins in your ELISAs down to the picogram levels Room temperature storage a consistent product with ambient shipping and no need to store at 4 C 8-hour working solution stability consistent performance of the working solution over an 8-hour period with only a 10% decrease in activity at 24 hours Flexible signal can be read in black or white opaque plates Emits light at 425 nm Immediate light generation 30-minute stable light output Net Relative Light Units (RLU) Thermo Scientific SuperSignal ELISA Pico Substrate Luminol-Based, Brand A Dioxetane-Based, Brand B Time (minutes) Working Solution Stability at Room Temperature -hour working solution stability 8-hour working solution stability Only 10% loss of activity after 24 hours Immediate and stable light generation with Thermo Scientific SuperSignal ELISA Pico Chemiluminescent Substrate. 200 pg of biotinylated HRP or biotinylated AP were added to separate wells of Thermo Scientific NeutrAvidin Coated White Polystyrene Plates. The plates were then incubated for 30 minutes at room temperature (RT) on a plate shaker and then each well was washed three times with Thermo Scientific BupH Tris Buffered Saline. Working solutions of chemiluminescent substrates were prepared according to the manufacturers instructions. For SuperSignal ELISA Pico Chemiluminescent Substrate and another luminol-based system (Brand A), 100 µl of each substrate working solution was added to the appropriate plate well. For the dioxetane-based system, wells were washed with 1X Assay Buffer. Next, 100 µl of the dioxetane working solution was added to the appropriate plate well. All plates were incubated on a plate shaker at RT for 1 minute. Plates were then read on a Dynex MLX* Luminometer with a 0.2 second read time per well. Several readings were taken over a 30-minute period. Bradley, K.A., et al. (2004). J. Biol. Chem. 278, McKevitt, M., et al. (2003). Genome Res. 13, SuperSignal ELISA Pico Chemiluminescent Substrate Includes: Luminol/Enhancer Stable Peroxide Buffer 3709 SuperSignal ELISA Pico Chemiluminescent Substrate Includes: Luminol/Enhancer Stable Peroxide Buffer Patent, see Patent Index. 100 ml 50 ml 50 ml 250 ml 125 ml 125 ml Additional dry ice and/or freight charges Additional hazardous handling charge *Trademark, see Trademark Index To order or for technical support, contact your local office or distributor. See pages -2,

56 ELISA Chemiluminescent Substrates SuperSignal ELISA Femto Maximum Sensitivity Substrate The most powerful substrate for high-throughput screening/ diagnostic applications with high sensitivity and superior low-end linearity. Thermo Scientific SuperSignal ELISA Femto Maximum Sensitivity Substrate is formulated for superior protein detection and low-end linearity in ELISA applications. SuperSignal* ELISA Femto Substrate s rapid signal generation has the added benefit of decreasing the substrate incubation period, generating detectable light within one minute of addition to trace amounts of soluble HRP, saving up to 30 minutes per assay. This feature is ideal for high-throughput screening (HTS) applications in which as many as 100,000 assays may be run daily on robotic equipment. Because incubation periods are often a limiting factor in the development of rapid automated assays, SuperSignal ELISA Femto Substrate is the logical choice for automated HTS applications. Immediate light generation intense signal generated immediately at both room temperature and 37 C Improved low-end linearity easy detection of low quantities of proteins with high signal:noise ratios and low-end linearity of dose response curves High sensitivity femtogram-level detection of target proteins Reduction in assay time high sensitivity allows for reduction in incubation steps Stability storage for six months at room temperature or a minimum of 12 months at 4 C with a six-hour working solution stability Emits light at 425 nm Net Relative Light Units (RLU) ,000 1,500 fg IL-2 Femtogram detection of target protein and superior low-end linearity. The dose response curve generated from an IL-2 ELISA illustrates the exceptional low-end linearity achieved with SuperSignal ELISA Femto Substrate and the incredible sensitivity, which was down to 18 fg of Interleukin-2. The R 2 value of the curve was 1.00 for signal generated at less than 1,00 fg of IL-2. Incubation time required to reach maximum signal. SuperSignal ELISA Femto Maximum Sensitivity Substrate SuperSignal ELISA Pico Chemiluminescent Substrate Dioxetane-based Substrate System Luminol-based Substrate System Acridan-based Substrate System TMB Colorimetric Substrate Net Relative Light Units (RLU) 1,500 1, minute at room temperature 1 minute at room temperature 30 minutes at room temperature 1 minute at room temperature 5 minutes at room temperature 15 minutes at room temperature Signal intensity and kinetics comparison of chemiluminescent substrates. The dose response curve using SuperSignal ELISA Femto Maximum Sensitivity Substrate in an IL-2 ELISA was compared to curves generated with a dioxetanebased substrate, another luminol-based substrate, an acridan-based system and TMB. SuperSignal ELISA Femto Maximum Sensitivity Substrate demonstrated immediate light generation with maximum peak intensity and high RLU values. Brandt, E.B., et al. (2003). J. Clin. Invest. 112, Hanley, N.R.S. and Hensler, J.G. (2002). J. Pharmacol. Exp. Ther. 300, Masri, H.P. and Cornellissen, C.N. (2002). Infect. Immun. 70, Su, S.V., et al. (2004). J. Biol. Chem. 279(18), pg IL SuperSignal ELISA Femto Maximum Sensitivity Substrate Includes: Luminol/Enhancer Stable Peroxide Solution SuperSignal ELISA Femto Maximum Sensitivity Substrate Includes: Luminol/Enhancer Stable Peroxide Buffer Patent, see Patent Index. Thermo Scientific SuperSignal ELISA Femto Substrate SuperSignal ELISA Pico Substrate Dioxetane-based Substrate Luminol-based Substrate Acridan-based Substrate 100 ml 50 ml 50 ml 250 ml 125 ml 125 ml 182 To order or for technical support, contact your local office or distributor. See pages -2, 3.

57 ELISA Fluorescent Substrates QuantaRed Enhanced Chemifluorescent HRP Substrate The most sensitive red-shifted chemifluorescent HRP substrate for ELISA. The Thermo Scientific QuantaRed Enhanced Chemifluorescent HRP Substrate is the most sensitive fluorescent ELISA substrate, providing sensitivity comparable to enhanced chemiluminescence. QuantaRed* Substrate uses patent-pending technology to increase the fluorescent yield and sensitivity of the reaction based on ADHP (10-acetyl-3,7- dihydroxyphenoxazine). The reaction product is resorufin, a soluble, highly fluorescent compound, with excitation/emission maxima at approximately 570/585 nm that can be measured in either fluorometric or colorimetric modes. The long wavelength emission minimizes interference from the low wavelength (blue and green) autofluorescence present in many biological samples. QuantaBlu Fluorogenic Peroxidase Substrates The ideal fluorescent substrates for use with peroxidase enzymes. Thermo Scientific QuantaBlu Substrate generates a nonbleaching blue fluorescent product upon reaction with peroxidase that does not photobleach. Fluorometric-based detection overcomes the limitations of colorimetric substrate detection, which does not allow for quantitation of greater than four optical density units. QuantaBlu* Substrate allows for stopped, nonstopped and kinetic assays to be performed. Incubation times for stopped and nonstopped assays can be varied between one to 90 minutes at either room temperature or 37 C. QuantaBlu Substrate exhibits a flat baseline in assays, which facilitates low-level detection sensitivity and allows for high signal:noise ratios. QuantaBlu Substrate enables rapid detection of peroxidase at very low concentrations. Peroxidase is detected at 0-10 pg per well from 1.5 to.5 minutes of substrate incubation time. At cycle 1 (1.5 minute incubation) as little as 2.5 pg of peroxidase could be detected, while at cycle (.5 minute incubation) 0.25 pg of peroxidase could be detected. More sensitive than TMB, OPD or ABTS substrates Flexible stopped, nonstopped or kinetic assays possible Large dynamic range (4 log peroxidase concentration range) Excellent stability working solution is stable for 24 hours Large stokes shift; excitation/emission maxima of 325/420; range of / Does not photobleach Sensitive low picogram to femtogram detection Red-shifted less interference from autofluorescence generated by biological samples Convenient compatible with existing filter sets, no need to purchase special filter sets Complete easy to use; no buffers to prepare or reagent powders to dissolve Flexible effective stop solution result in stable signal for at least 4 hours Versatile produces strong signal for easy fluorometric or colorimetric measurement Signal-to-noise Ratio QuantaRed Enhanced Chemifluorescent HRP Substrate Sufficient reagents to perform 10 x 9-well assays. Includes: ADHP Concentrate Enhancer Solution Stable Peroxide Solution Stop Solution Patent, see Patent Index ,000 2,000 3,000 4,000 Biotinylated-HRP, pg/well Kit 1 ml 50 ml 50 ml 10 ml Comparison of Thermo Scientific QuantaBlu Substrate to other substrates. QuantaBlu Substrate and the colorimetric substrates were incubated for 30 minutes at RT, followed by addition of a stop solution. QuantaBlu Substrate produced the greatest signal-to-noise ratios and exhibited the lowest detection limit. Ayala, P., et al. (2002). Infect. Immun. 70, Atamna, H., et al. (2000). Proc. Natl. Acad. Sci. USA 97, Donaldson, K.A., et al. (2004). Biosensors and Bioelectronics 20, Ingelsson, M. et al. (2004). Neurology 2, Jefcoat, A.M., et al. (2001). Am. J. Physiol. Lung Cell. Mol. Physiol. 281, L Savage, M.D., et al. (1998). Previews 2(1), -9. Savage, M.D., et al. (1998). Previews 2(2), Thermo Scientific QuantaBlu Substrate TMB ABTS OPD 1519 QuantaBlu Fluorogenic Peroxidase Substrate Kit Includes: Substrate 250 ml Stable Peroxide Solution 30 ml Stop Solution 275 ml 1512 QuantaBlu NS/K Substrate Kit (for nonstopped and kinetic assays) Includes: Substrate 250 ml Stable Peroxide Solution 30 ml Patent, see Patent Index. Additional dry ice and/or freight charges Additional hazardous handling charge *Trademark, see Trademark Index To order or for technical support, contact your local office or distributor. See pages -2,

58 ELISA Colorimetric Substrates Soluble Colorimetric Substrates for HRP Chromogenic ELISA subtrates result in a soluble, colored product whose intensity is easily measured with a spectrophotometric plate reader equipped with the appropriate lamps and filters. TMB (3,3', 5,5'-tetramethylbenzidine) is the most popular and versatile colorimetric substrate for ELISA methods involving horseradish peroxidase (HRP) enzyme conjugates. For example, TMB is the preferred substrate for most cytokine sandwich ELISA kits, including our own products (see chapter 12). Several formulations of TMB that differ in sensitivity and assay-specific behavior are available. ABTS (2, 2'-azinobis[3-ethylbenzothiazoline--sulfonic acid]- diammonium salt), like one of the TMB formulations, provides for accurate measurement in kinetic assays. OPD (o-phenylenediamine dihydrochloride) is another relatively sensitive HRP substrate that produces a yellow-orange color. Summary of Thermo Scientific ELISA Substrates for horseradish peroxidase. Kinetic Description Highlights Sensitivity Measurement Possible Absorbance Maximum Color ABTS Solution Single, ready-to-use solution Low Yes 405 nm Green Slow reaction that can be easily followed with a kinetic reader ABTS Tablets Can be easily dissolved in a substrate buffer such as Low Yes 405 nm Green Stable Peroxide Substrate Buffer (10X) (Product # 3402) OPD Can be easily dissolved in a substrate buffer such as High No 492 nm Orange Stable Peroxide Substrate Buffer (10X) (Product # 3402) TMB Substrate Kit Easy-to-use, two-component system Very High No 450 nm Yellow Results in seconds No DMF or DMSO in reagent Ultra TMB-ELISA Single, ready-to-use solution Very High No 450 nm Yellow Highest sensitivity No DMF or DMSO in the reagent Turbo TMB Single, ready-to-use solution High No 450 nm Yellow No DMF or DMSO in the reagent Slow TMB Single, ready-to-use solution Optimized for kinetic assays No DMF or DMSO in the reagent Medium Yes 450 nm Yellow ABTS Substrates ABTS (2,2 -azinobis [3-ethylbenzothiazoline--sulfonic acid]-diammonium salt) is a water-soluble HRP substrate that yields a green end product upon reaction with peroxidase. The green product has two major absorbance peaks, 410 nm and 50 nm. ABTS is less sensitive than OPD and TMB in ELISA applications. It is less readily oxidized, and its color development is slower (approximately 20 minutes). This may be advantageous if unacceptable background results from the use of the OPD or TMB substrates due to higher sensitivities. Paing, M.M., et al. (2002). J. Biol. Chem. 277, Sau-Ching Wu, S.-C., et al. (2002). Appl. Envir. Microbiol. 8, ABTS 50 tablets (10 mg/tablet) 3715 ABTS Substrate Solution 250 ml (Ready-to-use) 3402 Stable Peroxide Substrate Buffer (10X) 100 ml OPD Substrates OPD (o-phenylenediamine dihydrochloride) yields a water-soluble yellow-orange product when reacted with peroxidase with an absorbance maximum of 492 nm. OPD can easily be dissolved in a substrate buffer such as Thermo Scientific Stable Peroxide Substrate Buffer (Product # 3402). Buffered stable peroxide is used by researchers who prefer to make their own HRP-ELISA substrates. This is done easily by adding a chromogen of choice to the buffer. The Stable Peroxide Substrate Buffer has a long shelf life (12 months after receipt), and is provided as a 10X concentrate. A total volume of 1,000 ml can be made from one 100 ml bottle of concentrate. If you use 10 ml of substrate per plate, you can make substrate for 100 plates from only one bottle of Stable Peroxide Substrate Buffer OPD 25 g powder 3400 OPD Tablets 50 tablets (5 mg/tablet) 3402 Stable Peroxide Substrate Buffer (10X) 100 ml 184 To order or for technical support, contact your local office or distributor. See pages -2, 3.

59 ELISA Colorimetric Substrates Pierce TMB Substrate Solutions and Kit TMB (3,3,5,5 -tetramethylbenzidine) is a chromogen that yields a blue color (measurable at 370 nm or 52 nm) when oxidized with hydrogen peroxide (catalyzed by HRP). 1,2 The color then changes to yellow (measured at 450 nm) upon addition of sulfuric or phosphoric acid to stop the reaction. 1,2 TMB is very sensitive and more quickly oxidized than other HRP substrates, resulting in faster color development. 3 Thermo Scientific Pierce TMB Substrate Solutions are one-component substrates that require no preparation before use. Unlike other commercially available substrates, these products contain no DMF or DMSO. There are three formulations that differ primarily in their sensitivities. Slow TMB is intermediate in sensitivity ideal for kinetic readings. The sensitivity of the Turbo TMB compares to that of OPD used at approximately 1 mg/ml. Ultra TMB-ELISA produces the highest signal:noise ratio and sensitivity in the picogram range. Highlights of Pierce TMB Substrate Solutions: Ready-to-use single component No hydrogen peroxide required No filtering required Noncarcinogenic Various sensitivities to suit any assay Absorbance 450 nm (TMB) 410 nm (ABTS) x x 10-5 Turbo TMB Slow TMB ABTS.25 x x x 10-4 Dilution of HRP Conjugate (from 1 mg/ml stock) Comparison of sensitivities of Thermo Scientific Pierce Turbo TMB, Slow TMB and ABTS Substrates. Absorbance (450 nm) Thermo Scientific Pierce Ultra TMB-ELISA Substrate produces more signal than other TMB substrates. Signal-to-noise Ratios Concentration of Human IFN Gamma (pg/ml) Thermo Scientiic Pierce Ultra TMB ELISA Supplier B Supplier D Thermo Scientific Pierce Ultra TMB-ELISA Substrate produces higher signal:noise ratios than other TMB substrates. 1. Josephy, P.D., et al. (1982). J. Biol. Chem. 257(7), Josephy, P.D., et al. (1983). J. Biol. Chem. 258(9), Liem, H.H., et al. (1979). Anal. Biochem. 98, Hong, P.W., et al. (2002). J. Virol. 7, Murphy, M.B., et al. (2003). Nucleic Acids Res. 31, e110. Su, S.V., et al. (2004). J. Biol. Chem. 279(18), Tek, V. and Zolkiewski, M. (2002). Protein Sci. 11, Thomas, P.E., et al. (197). Anal. Biochem. 75, Weimer, B.C., et al. (2001). Appl. Envir. Microbiol. 7, Wu, S.-C. and Wong, S.-L. (2002). Appl. Envir. Microbiol. 8, Supplier I Supplier K TMB Sources Used Supplier M Slow TMB Substrate Solution 250 ml Turbo TMB Substrate Solution 250 ml Ultra TMB-ELISA Substrate Solution 250 ml TMB Substrate Kit Kit Includes: Peroxidase Substrate (TMB) 200 ml Peroxide Solution (H ) 200 ml Thermo Scientific Pierce Ultra TMB-ELISA Supplier B Supplier D Supplier I Supplier K Supplier M Supplier N Supplier N Additional dry ice and/or freight charges Additional hazardous handling charge *Trademark, see Trademark Index To order or for technical support, contact your local office or distributor. See pages -2,

60 ELISA Colorimetric Substrates Soluble Colorimetric Substrates for AP The most common ELISA substrate for alkaline phosphatase (AP) is the chromogen p-nitrophenyl phosphate (PNPP), which is available in several formats. PNPP yields a yellow reaction product that is water- soluble and absorbs light at 405 nm. Powder or single-use tablets of PNPP must be dissolved and reacted in diethanolamine buffer. Stable solutions of PNPP provide added convenience and reproducibility. PNPP Substrates Used to detect alkaline phosphatase label in ELISA applications. PNPP (p-nitrophenyl phosphate, disodium salt) is a widely used substrate for detecting alkaline phosphatase in ELISA applications. 1 When alkaline phosphatase and PNPP are reacted, a yellow watersoluble reaction product is formed. This product absorbs light at 405 nm. Thermo Scientific PNPP is available in four formats. Dry PNPP is available either as a crystalline powder or 5 mg tablets. Also available is the Phosphatase Substrate Kit, which contains PNPP tablets and a Diethanolamine Buffer to yield more than 500 ml of substrate. The Diethanolamine Substrate Buffer is also provided individually as a 5X concentrate; the formulation has an optimal and consistent ph and it is stable even at a 1X concentration. Until recently, the method for preparing PNPP solutions required dissolving the powder or tablets in buffer and then diluting the solution to the desired concentration. Substrate instability made it necessary to prepare it on a daily basis. Our PNPP Substrate Solution circumvents this time-consuming and variable-introducing step by providing a single-component PNPP substrate. This substrate is stable for 12 months at 2-8 C and can be stopped with conventional methods. Soluble Colorimetric Substrate for Beta-Galactosidase When using β-galactosidase as the label for proteins in ELISA studies, a wide variety of substrates are available, including o-nitrophenyl-β-dgalactopyranoside (ONPG), naphthol-as-bi-β-d-galactopyranoside (Nap-Gal) and 4-methyl-umbelliferyl-β-D-galactopyranoside (Mum-Gal). However, it is important to choose a substrate with adequate solubility, that uses readily available equipment and that gives a significant Reference 1. Snyder, S.L., et al. (1972). Biochim. Biophys. Acta 258, Bosque, P.J., et al. (2002). Proc. Natl. Acad. Sci. USA 99, Jan, J.-T., et al. (2000). J. Virol. 74, PNPP Sufficient to prepare 25 L of substrate when diluted in Diethanolamine Substrate Buffer PNPP Tablets Sufficient to prepare 525 ml of substrate when diluted in Diethanolamine Substrate Buffer Phosphatase Substrate Kit Sufficient reagents for 525 ml of substrate. Includes: Diethanolamine Buffer PNPP Tablets 25 g powder 105 tablets (5 mg/tablet) reading over the background. ONPG is a superior β-galactosidase substrate option. The product formed is completely soluble and has a high extinction coefficient at 405 nm. The substrate yields a yellow product that is easily detectable in the visual range after stopping the reaction with 1 M sodium carbonate. Kit 225 ml 105 tablets (5 mg/tablet) 3721 PNPP Substrate Solution 100 ml 3404 Diethanolamine Substrate Buffer (5X) 225 ml ONPG Substrate For detection of β-galactosidase label in ELISA applications. ONPG (o-nitrophenyl-β-d-galactopyranoside) is the preferred colorimetric substrate for ELISA applications involving β-galactosidase (β-gal) reporter enzyme. The enzyme-substrate reaction yields a yellow product with an absorbance maximum at 420 nm but whose intensity is also very high at 405 nm (matching a common plate reader filter set). Reference Craven, G.R., et al. (195). J. Biol. Chem. 240, ONPG 5 g powder 18 To order or for technical support, contact your local office or distributor. See pages -2, 3.

61 Immunohistochemistry Substrates Immunohistochemistry Immunohistochemistry (IHC) combines anatomical, immunological and biochemical techniques for the identification of specific tissue components by means of a specific antigen/antibody reaction tagged with a visible label. IHC makes it possible to visualize the distribution and localization of specific cellular components within a cell or tissue. The term immunohistochemistry is often used interchangeably with immunocytochemistry and immunostaining. The principle of immunohistochemistry has existed since the 1930s 1, but it was not until 1942 that the first immunohistochemistry study was reported. Coons et al. 2 used FITC-labeled antibodies to localize Pneumococcal antigens in infected tissues. Since then improvements have been made in protein conjugation, tissue-fixation methods, detection labels and microscopes, making immunohistochemistry a routine and essential tool in many laboratories. Typical immunohistochemistry protocol. 1. Fix tissue to be stained. 2. Block nonspecific sites with serum or blocker protein. 3. Incubate with primary antibody (1:100-1:1,000). 4. Drain reagent. 5. Incubate with secondary antibody-enzyme conjugate (1:2,000-1:5,000).. Wash. 7. Incubate with substrate. 8. Visualize the stained tissue. Thermo Scientific Immunohistochemistry Kits and Reagents Normal Sera for Blocking The most popular blocking agent for immunohistochemical staining; great for use as blocking reagents or negative controls Normal Goat Serum 2 ml Normal Goat Serum 10 ml Normal Horse Serum 2 ml 3187 Normal Human Serum 2 ml Normal Mouse Serum 2 ml Today, the most popular methods of detection are with enzyme antibody and fluorophore-antibody conjugates. Methods utilizing enzyme conjugated antibodies were developed independently by Nakane and Pierce 3 and Avrameas and Uriel. 4 After the antigen-antibody reaction, the enzyme label is reacted with a substrate to yield an intensely colored product that can be analyzed with an ordinary light microscope. A further advantage of using enzyme labeled systems is the option to make the product electron dense for electron microscopy. Thermo Scientific Products include traditional reagents for IHC as well as fluorescent probes and high-content screening (HCS) reagents and kits that comprise state-of-the-art advancements in IHC. Guide to Thermo Scientific IHC-related Products. Product Group Location in Catalog Avidin-Biotin Complex (ABC) Staining Next Page DAB and other colorimetric staining reagents Pages that follow Fluor-conjugated secondary antibodies Page 134 this section Fluorescent labeling kits Catalog Section 9 High-Content Screening (HCS) Reagents Kits Catalog Section Marrack, J. (1934). Nature 133, Coons, A.A., et al. (1942). J. Immunol. 45, Nakene, P.K. and Pierce, G.B. (19). J. Histochem. Cytochem. 92, Avrameas, S. and Uriel, G. (19). C.R. hebd. Seanc. Acad. Sci. (D) Paris 22, Normal Mouse Serum 5 ml Normal Rabbit Serum 2 ml Normal Rabbit Serum 5 ml Normal Rat Serum 2 ml Normal Swine Serum 2 ml Formaldehyde Ampules, Methanol-free High-purity, 1% (w/v) formaldehyde for use as crosslinker and fixative. Formaldehyde is a highly reactive, cell-permeable agent that is used by researchers as a reversible crosslinking agent for proteins and nucleic acids within the cell or as a general cell-fixing agent for imaging-based applications. Cell permeability allows the reagent to readily enter living cells enabling intracellular applications, such as protein interaction discovery or bioimaging. Formaldehyde produces reversible crosslinks with protein and nucleic acids by coupling primary amines that are within proximity. Crosslinks can be reversed simply by heating the sample. Pure and stable prepared directly from pure granular paraformaldehyde and sealed in ampules under an inert atmosphere, ensuring that it is methanol-free and resistant to oxidation Versatile use to crosslink molecules in protein-protein and proteinnucleic acid interaction discovery, bioimaging and flow cytometry Aliquot packaging choose 1 ml or 10 ml ampules suitable as aliquots for low- and high-volume applications Convenient starting concentration 1% (w/v) solution simplifies dilution to the required working concentration % Formaldehyde (w/v), Methanol-free 10 x 1 ml % Formaldehyde (w/v), Methanol-free 10 x 10 ml Additional dry ice and/or freight charges Additional hazardous handling charge *Trademark, see Trademark Index To order or for technical support, contact your local office or distributor. See pages -2,

62 Immunohistochemistry Substrates ABC Staining Kits Systems that offer highly sensitive and rapid detection. Thermo Scientific ABC Staining Kits for the avidin-biotin complex (ABC) technique are highly sensitive, produce very low background staining and have rapid avidin-biotin interactions. Highly diluted primary antibodies can be used with ABC Staining Kits, producing comparable stain intensity to other methods that require higher concentrations of antibody. Kits with a secondary antibody are selected by the species of primary antibody to be used. For example, if the primary antibody (IgG) is produced in mice, select the ABC Mouse IgG Kit. All you need to create a sensitive detection system is your specific primary antibody, an ABC Kit with biotinylated antibody and a substrate for horseradish peroxidase. The Standard Kits include only the avidin and biotinylated enzyme. They are useful if an ABC Kit is not available for your specific species of primary antibody or if the primary antibody is directly labeled with a Thermo Scientific EZ-Link Biotinylation Reagent (see Catalog Section 9: Protein Labeling). The biotinylated antibody for your specific application can then be used, along with blocking serum and a Standard ABC Kit. The Ultra-Sensitive ABC Peroxidase Staining Kits are more sensitive than the ABC Peroxidase Staining Kits, without exhibiting increased background staining. These kits supply the extra sensitivity needed for localizing antigens present in very small quantities. An expensive primary antibody may be diluted approximately five-fold higher than with the standard ABC Peroxidase Kit while producing equal staining intensity. 1 B B B A B E Substrate E B B A B E E B Thermo Scientific Substrates to use with horseradish peroxidase ABC kits. ELISA TMB Substrate Kit Product # Turbo TMB Product # ABTS Product # 3715 Colorimetric Western Blotting Chloronaphthol Product # TMB-Blotting Product # Chemiluminescent Western Blotting SuperSignal West Pico Substrate Product # SuperSignal West Dura Substrate Product # SuperSignal West Femto Substrate Product # Histochemical Metal Enhanced DAB Substrate Kit Product # 3405 Reference 1. Bayer, E.A., et al. (1988). Anal. Biochem. 170, Product # ABC Staining Kit Kit Ultra-Sensitive ABC Peroxidase Mouse IgG Staining Kit Includes: Biotinylated Anti-Mouse IgG Antibody Blocking Buffer Avidin Biotinylated HRP Ultra-Sensitive ABC Standard Peroxidase Kit Rabbit IgG Staining Kit Includes: Biotinylated Anti-Rabbit IgG Antibody Blocking Buffer Avidin Biotinylated HRP ABC Standard Peroxidase Staining Kit Kit Includes: Avidin Biotinylated HRP Ultra-Sensitive ABC Standard Peroxidase Kit Staining Kit Includes: Avidin Biotinylated HRP Note: Ultra-Sensitive ABC Kits allow the primary antibody to be diluted approximately five-fold higher than with regular ABC kits. Substrate B Avidin-biotin complex (ABC) for signal amplification. 188 To order or for technical support, contact your local office or distributor. See pages -2, 3.

63 Immunohistochemistry Substrates Metal Enhanced DAB Substrate Kit The most sensitive DAB substrate formulation available. The color produced by DAB (3,3'-diaminobenzidine) can be intensified by the addition of metals such as nickel, copper, silver and cobalt that form more dense complexes upon reaction in HRP-peroxide substrate reactions. The Thermo Scientific Pierce Metal Enhanced DAB Substrate Kit optimizes this intensifying chemistry, producing an exceptionally sensitive colorimetric detection system for Western blot, dot-blot and immunohistochemistry applications. The near-black signal (precipitate) is easy to photograph. Incredible sensitivity 50 times more sensitive than the traditional DAB method and 30 times more sensitive than other metal-intensified versions of DAB! Low background, high intensity get a crisp dark brown-black precipitate and, even with the increased intensity, background is almost nonexistent! Six-hour stability working solution is stable for more than six hours at room temperature! Other DAB substrates must be used immediately Comparison of DAB substrates and methods. Substrate Lower Detection Limit 3 DAB Substrate (Method of Graham and Karnovsky 1 ) Metal Enhanced DAB Substrate (Method of Adams 2 ) Metal Enhanced DAB Substrate Kit (Pierce Product # 3405) Pierce Peroxidase Detection Kit ng Horseradish peroxidaseconjugated streptavidin ng Horseradish peroxidaseconjugated streptavidin ng Horseradish peroxidaseconjugated streptavidin The benefits of metal-enhanced DAB in a complete kit. This immunohistochemical staining kit bundles the Thermo Scientific Pierce Metal Enhanced DAB Substrate Kit with all necessary components to stain, counter-stain and preserve experimental results with frozen or paraffin tissue section. The Thermo Scientific Pierce Peroxidase Detection Kit eliminates the need to procure numerous reagents from various sources, resulting in a time- and cost-effective way to perform peroxidase-based tissue staining. Harris Hematoxylin nuclear stain and mounting medium allow counter-staining and tissue preservation, respectively. The detection method is compatible with direct or avidin-biotin-complex (ABC) probing strategies. A. Metal enhanced DAB substrate staining of GFAP. B. Negative control (no primary antibody). Thermo Scientific Pierce Metal Enhanced DAB Ordinary DAB Method (Graham and Karnovsky, 19) Superior staining performance with Thermo Scientific Pierce Metal Enhanced DAB Substrate Kit. Specific staining of prostatic acid phosphatase detected with the Pierce Kit (left panel) exhibits higher intensity and greater resolution than staining detected by non-enhanced DAB method (right panel). 1. Graham, R.C. and Karnovsky, M.J. (19). J. Histochem. Cytochem. 14, Adams, J.C. (1981). J. Histochem. Cytochem. 29, Rodbard, D. (1978). Anal. Biochem. 90, Adams, J.C. (1977). Neuroscience 2, Geoghegan, W.D. and Ackerman, G.A. (1977). J. Histochem. Cytochem. 25, Metal Enhanced DAB Substrate Kit Kit Includes: 10X Metal Enhanced DAB 25 ml Stable Peroxide Buffer 250 ml Applications: The detection of peroxidase in tissue immunohistostaining applications using the metal-enhanced DAB precipitating substrate DAB staining of fixed paraffin sections, smears and frozen section preparations Tissue or cell staining 3000 Pierce Peroxidase Detection Kit Kit Includes: DAB/Metal Concentrate (10X) 25 ml Peroxidase Detection Reagent Pack: Stable Peroxide Substrate Buffer (1X) 250 ml Universal Blocker in TBS 250 ml Peroxidase Suppressor 2 x 100 ml BupH Tris Buffered Saline 4 packs Surfact-Amps 20 (10% Tween*-20) 10 ml Harris Modified Hematoxylin 100 ml (without Acetic Acid, Hg free) Mounting Medium (dropper bottle) 0 ml Staining of glial fibrillary acidic protein (GFAP) using the Thermo Scientific Pierce Peroxidase Detection Kit. Panel A. Staining was performed as indicated in the protocol provided with the kit. Anti-GFAP and goat anti-mouse IgG biotin conjugate were used as the primary and secondary antibodies, respectively. Streptavidin-HRP was the detection conjugate. Panel B. Same as Panel A except no anti-gfap primary antibody was used. Additional dry ice and/or freight charges Additional hazardous handling charge *Trademark, see Trademark Index To order or for technical support, contact your local office or distributor. See pages -2,

64 Immunohistochemistry Substrates and Counterstain NBT/BCIP Substrate Solutions The combination of NBT and BCIP is an ideal system for blotting or staining applications with alkaline phosphatase (AP). Together, they yield an intense, black-purple precipitate that provides much greater sensitivity than either substrate alone. This reaction proceeds at a steady rate, allowing accurate control of its relative sensitivity. Thermo Scientific NBT/BCIP Substrate characteristically produces sharp band resolution with minimal background. Ready-to-use, single-component solutions Regular formulation ideal for Western blotting Suppressor formulation contains levamisole for inhibition of endogenous enzyme, making it ideal for immunohistochemistry applications Reference Stuyver, L.J., et al. (2003). Antimicrob. Agents Chemother. 47, NBT/BCIP Substrate Solution 250 ml NBT/BCIP Plus Suppressor Substrate Solution 100 ml Colorimetric β-galactosidase Precipitating Substrates For detection of β-galactosidase in immunohistochemical and clone expression applications. X-Gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside) is a chromogenic substrate for β-galactosidase. It yields a blue precipitate, is soluble in dimethylformamide, and can be used to detect vectors, plasmids or DNA fragments that contain the β-galactosidase gene. IPTG (isopropyl-β-d-thiogalactopyranoside) is used to maximize the expression of β-gal-fused genes in expression vectors. Peroxidase Suppressor A stable, easy-to-use endogenous peroxidase inhibitor. The immunoperoxidase method is commonly used for immunostaining of cells and tissues. However, the analysis is often complicated by the presence of endogenous peroxidase and pseudo-peroxidase activity in the cells and tissues. This results in nonspecific staining when a substrate such as diaminobenzidine (DAB) is present. Nonspecific staining is difficult to distinguish from the immunochemical marker, making it difficult to distinguish the final location of the antigen of interest. The Thermo Scientific Peroxidase Suppressor inhibits endogenous peroxidase activity even more effectively than the method using hydrogen peroxide in methanol. Reference Maniatis, T., et al. (1982). Molecular Cloning: A Laboratory Manual. New York: Cold Spring Harbor Laboratory X-Gal 100 mg powder 3400 IPTG 1 g powder Reference Charter, N.W. (2002). J. Biol. Chem. 277, Peroxidase Suppressor Supplied in methanol solution. 100 ml DAPI Counterstaining Reagents Compatible blue color with fluorescent staining. Conventional dyes cannot be used in immunofluorescent-stained tissues because they often contribute to background fluorescence. However, DNA marker dyes such as bisbenzimide (Hoechst 33258) and DAPI stain a pale blue when viewed with a filter set in the true blue emission range. DAPI counterstain for DNA content is compatible with fluorescein and rhodamine probes ,-Diamidino-2-phenylindole, hydrochloride (DAPI) 10 mg 190 To order or for technical support, contact your local office or distributor. See pages -2, 3.

65 Immunohistochemistry Fluorescent Imaging Cell Imaging Technologies Fluorescence Microscopy and High-Content Analysis Traditional immunohistochemical staining methods are rapidly being replaced by fluorescence imaging techniques. The development and availability in recent years of high-performance fluorescent dyes that have complementary (non-overlapping) emission spectra enables simultaneous detection and analysis of multiple parameters in whole cells. The basic instrument for detection of fluorescent-stained cells is the fluorescence microscope. High-content screening and analysis (HCS or HCA) refers to the use of fluorescence microscopy for automated, digital image-capture (usually from 9-well plates) and statistical analysis of the multi-parametric data with specialized software. Thermo Scientific Products include a full range of fluorescent dyes and stains, labeled primary and secondary antibodies, general buffers and reagents, target-specific HCS reagent kits, imaging instrumentation and analysis software, and fluorescent reporter cell lines. Fluorescent Dyes and Conjugates Historically, fluorescein and rhodamine, together with the fluorescent stains Hoechst and DAPI, have been the most popular fluorophores for labeling antibodies and other biomolecules for fluorescence microscopy. In recent years, the growing demand for multiplex assays and high throughput have driven the development of many new fluorescent dyes that are brighter, more photostable, and comprise a broader range of nonoverlapping spectra. Thermo Scientific DyLight Fluors include nine, high-performance fluorescent dyes with emission spectra evenly distributed between the blue and infrared range ( nm). DyLight* Fluors are available in conjugated form with secondary antibodies and streptavidin (page 134) and in activated form for labeling (page 302). Traditional fluorescein and rhodamine conjugates and labeling reagents are also available (pages ; ). Primary antibodies are listed in Section 12. Thermo Scientific DyLight Fluor Wavelength (nm) General Reagent Kits for HCS The Cellomics Toolbox Kits (page 417) include general buffers for staining, nuclear and whole cell stains, and conjugated secondary antibodies that can be used in conjunction with a user-supplied primary antibody to perform multiparameter, target-specific immunofluorescence-based assays. Choose either the Rabbit or Mouse Antibody Detection WCS Toolbox Kits, which contain DyLight 549 conjugated secondary antibodies for detecting target-specific primary antibodies. Complete, Target-Specific HCS Kits Cellomics High-Content Screening (HCS) Reagent Kits (page ) are powerful tools for cell-based quantitative imaging and analysis of specific molecular targets and cellular responses. Cellomics HCS Reagent Kits are designed and validated with the Thermo Scientific ArrayScan HCS Reader and BioApplication Software, together providing a Total Solution for high-content screening. HCS Reagent Kits are also fully compatible with other fluorescence-based HCS platforms and conventional fluorescence microscopy. Control Etoposide Fluorescence analysis of DNA damage and repair. A549 cells were not treated (left) and treated (right) with 50 µm etoposide for 3 hours and then probed with Cellomics phospho-atm Activation Kit (page 412). The nuclei (blue) were detected with Hoechst Dye. Phospho-ATM (red) was detected with a specific primary antibody and DyLight 549 Conjugated Secondary Antibody. Induction of DNA damage by etoposide leads to phosphorylation of ATM, especially in the nucleus. Using Cellomics BioApplication Software, etoposide concentration can be plotted against the difference between nuclear and cytoplasmic intensities for phospho-atm to calculate a dose response curve (EC 50 = 7.5 ± 1.1 µm). Instrumentation and Analysis Software ArrayScan* Instruments and BioApplication Software Modules are powerful, indispensable tools for detection and analysis of cell-based fluorescent data. The instrument provides automated, multi-channel fluorescence imaging for cells grown in 9-well plates. The BioApplication Software includes a powerful suite of analysis modules for statistical analysis of multiparameter fluorescent image data. See page for examples of data analysis with these tools. For more information on these products, visit Emission spectra of Thermo Scientific DyLight Fluors. Conjugated secondary antibodies and activated dyes for protein labeling are available for these highperformance fluorophores. Whole Cell Stains Whole cell stains detect and delineate the surface and general contents of cells by binding or reacting to primary amines or other functional groups that are present in all proteins. Thermo Scientific Cellomics Whole Cell Stains are available in four different colors and provide excellent staining of fixed cells for HCS assays and fluorescence microscopy (page 41). These stains are intense, highly photostable and compatible with standard fluorescence instrumentation. Fluorescent Reporter Protein Cell Lines Thermo Scientific Redistribution Technology is a cell-based assay platform that uses protein translocation as a readout for the activity of cellular signaling pathways. Redistribution Assays are high-throughput, high-content, cell-based assays primarily used for profiling of lead series, primary screening of compound libraries, and as readouts for gene-silencing studies using sirna. Redistribution Assays are based on labeling targets with fluorescent proteins (e.g., green fluorescent protein, GFP), generation of stably transfected assay cell lines, measurement of protein translocation and image analysis and quantification of cellular responses. For more information on Redistribution Assays, visit Additional dry ice and/or freight charges Additional hazardous handling charge *Trademark, see Trademark Index To order or for technical support, contact your local office or distributor. See pages -2,

66 Nucleic Acids EMSA ThermoScientific Nucleic Acid Detection Products LightShift Chemiluminescent EMSA Kit Identifies regulatory sequences and determines protein:dna binding regions and affinity. The Thermo Scientific LightShift Chemiluminescent EMSA Kit is an extraordinarily robust and sensitive system for performing electrophoretic mobility shift assays (EMSAs) to identify and characterize protein:dna binding interactions. The kit includes reagents for setting up and customizing protein:dna binding reactions, a control set of DNA and protein extract to test the kit system, stabilized streptavidin-hrp conjugate to probe for the biotinlabeled DNA target, and an exceptionally sensitive chemiluminescent substrate module for detection. The principle for LightShift* EMSA Detection is similar to a Western blot. Biotin end-labeled duplex DNA is incubated with a nuclear extract or purified factor and electrophoresed on a native gel. The DNA is then rapidly (30 minutes) transferred to a positive nylon membrane, UVcrosslinked, probed with streptavidin-hrp conjugate and incubated with the substrate. The protocol from labeling to results can be accomplished in a single day (see Figure). All you need to perform the assay are purified DNA target that has been end-labeled with biotin, the protein extract you wish to test, nylon membrane and basic electrophoresis equipment. DNA targets may be synthesized with 5 or 3 biotin labels or they may be labeled after synthesis using the Thermo Scientific Biotin 3 End DNA Labeling Kit (see Product # on the following page). Nuclear, cytosolic or whole cell protein extracts may be obtained by a variety of methods, including the Thermo Scientific NE-PER Nuclear and Cytoplasmic Extraction Reagent Kit (Product # 78833). Includes EBNA control system to help new users develop a working assay and understand the methods used to confirm binding interaction specificity Excellent for detecting low-abundance proteins in nuclear extracts Sensitivity that surpasses radioactive and digoxigenin methods Compatible with previously established binding conditions for popular DNA:protein interactions Labeling minutes Reaction UV Cross-link Pre-run Gel Gel Transfer Detection 20 minutes 0 minutes 30 minutes 0-75 minutes Total Time (including prep time) = hours Extract Competitor Chemiluminescent EMSA of four different DNA:protein complexes. Biotin-labeled target duplexes ranged in size from bp. The EBNA reactions were supplemented with 2.5% glycerol and 0.05% NP-40, and the AP1 reactions were supplemented with 10% glycerol. The source of the Oct-1, AP1 and NF-κB transcription factors was a HeLa nuclear extract. EBNA-1 extract is provided as a control in the kit. Unlabeled specific competitor sequences (where used) were present at a 200-fold molar excess over labeled target. X-ray film exposure times for each system ranged from 2 minutes for EBNA, Oct-1 and AP1, and 5 minutes for NF-κB. Cornelussen, R.N.M., et al. (2001). J. Mol. Cell Cardiol. 33, Ishida, A., et al. (2002). J. Biol. Chem. 277(29), MacLachlan, T.K. and El-Deiry, W.S. (2002). Proc. Natl. Acad. Sci. USA 99(14), Matata, B.M. and Galinanes, M. (2002). J. Biol. Chem. 277(3), Sauzeau, V., et al. (2003). J. Biol. Chem. 278(11), Tarumi, T., et al. (2002). J. Biol. Chem. 277(21), EBNA Oct-1 API NF-κB LightShift Chemiluminescent EMSA Kit Kit Sufficient components for 100 binding reactions and detection reagents for ~800 cm 2 of membrane. Includes: 10X Binding Buffer Biotin-EBNA Control DNA Unlabeled EBNA DNA EBNA Extract Poly(dI dc) 50% Glycerol 1% NP-40 1 M KCl 100 mm MgCl mm EDTA, ph 8.0 5X Loading Buffer Stabilized Streptavidin-Horseradish Peroxidase Conjugate Luminol/Enhancer Solution Stable Peroxide Solution Blocking Buffer 4X Wash Buffer Substrate Equilibration Buffer 1 ml 50 µl 50 µl 125 µl 125 µl 500 µl 500 µl 1 ml 500 µl 500 µl 1 ml 1.5 ml 80 ml 80 ml 500 ml 500 ml 500 ml Patent, see Patent Index. Related Products Page Biotin 3 End DNA Labeling Kit NE-PER Nuclear and Cytoplasmic 11 Extraction Reagents Timeline for the Thermo Scientific LightShift EMSA Kit protocol. 192 To order or for technical support, contact your local office or distributor. See pages -2, 3.

67 Nucleic Acids Primer Labeling Biotin 3 End DNA Labeling Kit A complete kit for labeling the 3 end of DNA with biotin. The Thermo Scientific Biotin 3 End DNA Labeling Kit uses terminal deoxynucleotidyl transferase (TdT) to catalyze nontemplate-directed nucleotide incorporation onto the 3 -OH end of DNA. 1,2 TdT exhibits a substrate preference of single-stranded DNA, but it will label duplex DNA with 3 overhangs and blunt duplexes, albeit with a lower efficiency. 3 The Biotin 3 End DNA Labeling Kit has been optimized to incorporate 1-3 biotinylated ribonucleotides (biotin-11-utp) onto the 3 end of DNA strands. This labeling strategy has the advantage of localizing the biotin to the 3 end of the probe where it will be less likely to interfere with hybridization or sequence-specific binding of proteins. Biotin-labeled DNA probes can be used to facilitate non-isotopic detection in a variety of applications including electrophoretic mobility shift assays (EMSA), Northern or Southern blots, colony hybridizations, or in situ hybridizations. Non-isotopic labeling eliminates the hassle of hazardous radioactive materials or difficult-to-dispose-of waste 1-3 biotinylated ribonucleotides onto the 3 end of DNA strands for less interference with hybridization or sequence-specific binding of proteins Biotin-labeled probes are stable for more than one year 30-minute labeling procedure is fast and efficient n+2 n+1 n S A S A S A S A S A A A A n+2 n+1 n s = sense strand n+2 n+1 n a = antisense strand n+2 n+1 n Sequencing gel analysis of labeling efficiency. Ten different oligos (ranging in size from nt) were labeled using the Biotin 3 End DNA Labeling Kit. The products from the TdT reaction were then radiolabeled using T4 polynucleotide kinase (PNK) and [γ- 32 P]ATP. The PNK reactions were run on a 20% acrylamide/8 M urea/tbe. The position of the starting oligo (no biotin) is denoted by n. Incorporation of biotinlabeled ribonucleotide by TdT is limited to one or two incorporations per strand (positions n+1 and n+2, respectively). Labeling efficiencies ranged from 72% (EBNA sense strand) to 94% (Oct-1 sense strand). The kit control oligo labeled with 88-94% efficiency. n+2 n+1 n n+2 n+1 n Extract Competitor EBNA Oct-1 API SPI NF-κB NF-κB (TNF-α induced) (uninduced) EMSA results using 3 biotin-labeled duplexes. The sense and antisense strands were labeled using the Biotin 3 End DNA Labeling Kit and hybridized for 4 hours at room temperature to form duplexes containing the binding sites for the indicated transcription factors. Gel shift assays were performed using the Thermo Scientific LightShift Chemiluminescent EMSA Kit using 20 fmol duplex per binding reaction. The source of the transcription factors was a HeLa nuclear extract prepared using Thermo Scientific NE-PER Nuclear and Cytoplasmic Extraction Reagents (Product # 78833) (2 µl or -7 µg protein per reaction). In the case of the NF-κB system, nuclear extracts were made from HeLa cells that had been a induced with TNFα or cells that were untreated. Competition reactions containing 200-fold molar excess of unlabeled duplex were performed to illustrate the specificity of the protein:dna interactions. 1. Roychoudhury, R. and Wu, R. (1980). Methods Enzymol. 5, Roychoudhury, R., et al. (197). Nucleic Acids Res. 3, Michelson, A.M. and Orkin, S.H. (1982). J. Biol. Chem. 257, Cornelussen, R.N.M., et al. (2001). J. Mol. Cell Cardiol. 33, Biotin 3 End DNA Labeling Kit Sufficient components for 20 labeling reactions. Includes: 5X TdT Reaction Buffer Terminal Deoxynucleotidyl Transferase (TdT) Biotin-11-UTP Unlabeled Control Oligo Biotin-Control Oligo Kit 1 ml 50 µl 100 µl 140 µl 40 µl Related Products Page LightShift Chemiluminescent EMSA Kit NE-PER Nuclear and Cytoplasmic 11 Extraction Reagents 7701 Biodyne* B Nylon Membranes 145 Patent, see Patent Index. Additional dry ice and/or freight charges Additional hazardous handling charge *Trademark, see Trademark Index To order or for technical support, contact your local office or distributor. See pages -2,

68 Nucleic Acids Northern and Southern Blotting North2South Chemiluminescent Hybridization and Detection Kit Non-isotopic assay delivers sensitivity, consistency and flexibility. The Thermo Scientific North2South Hybridization and Detection Kit combines a novel enhanced luminol substrate for horseradish peroxidase (HRP) with optimized pre-made hybridization and blocking buffers to ensure consistent results and high sensitivity. Any nucleic acid blot can be hybridized with the appropriate biotinylated (biotin-labeled) oligonucleotide probe. After a brief membrane blocking step, the biotin label is detected with streptavidin-hrp and a 5-minute incubation with the chemiluminescent substrate. Results are easily recorded by brief exposure to X-ray film (1-15 minutes) or CCD camera. Detection of biotinylated DNA on a Southern blot using the Thermo Scientific North2South System and a cooled CCD imaging system. A Southern blot of biotinylated DNA samples from the Edvotek DNA Fingerprinting III Kit was detected using the North2South System and imaged for one minute using a cooled CCD camera system. Sensitivity equal to or greater than 32 P Amenable to many formats High level of light output and consistent low background allows for very short exposure times using cooled CCD camera systems or film Ready-to-use buffers and short process time makes switching from radioactive detection to chemiluminescent detection an easy transition, even for the novice user North2South DNA Probe Biotinylation Kit A safe alternative to radioactive labeling. The Thermo Scientific North2South Biotin Random Prime DNA Labeling Kit is based on the procedure of Feinberg and Vogelstein. Random heptanucleotides containing all possible sequences are annealed to a denatured DNA template. These act as primers for complementary strand synthesis by DNA polymerase. Biotinylated dntps in the reaction mix are incorporated into the newly synthesized DNA. This protocol yields biotin-labeled DNA of high activity for use as probes in hybridization experiments such as Southern and Northern hybridizations. The resulting hybrids can be detected with streptavidin-horseradish peroxidase (HRP). Ultimate sensitivity labels with biotinylated dutp, maximizing streptavidin-hrp binding and increasing sensitivity Works with as little as 100 ng starting DNA template Exonuclease-free Klenow fragment produces higher yields Each reaction yields probe sufficient for approximately three (10 x 10 cm) blots Adilakshmi, T. and Laine, R.O. (2002). J. Biol. Chem. 277(), Bach, S., et al. (2002). Infect. Immun. 70(2), Borst, E-M., et al. (1999). J. Virol. 73(10), Haertel-Wiesmann, M., et al. (2000). J. Biol. Chem. 275(41), North2South Chemiluminescent Kit Hybridization and Detection Kit Sufficient reagents for 1,000 cm 2 of membrane. Includes: Stabilized Streptavidin-Horseradish Peroxidase Conjugated Luminol/Enhancer Solution Stable Peroxide Solution Hybridization Buffer (2X) Hybridization Stringency Wash Buffer Blocking Buffer (4X) Wash Buffer Substrate Equilibration Buffer 1.5 ml 80 ml 80 ml 125 ml 375 ml 500 ml 500 ml 500 ml North2South Complete Biotin Random Kit Prime Labeling and Detection Kit Combines all the components of Product # and North2South Chemiluminescent Substrate for HRP Sufficient reagents for 1,000 cm 2 of membrane. Includes: Luminol/Enhancer Solution Stable Peroxide Solution 100 ml 50 ml 50 ml North2South Hybridization Buffer 125 ml North2South Hybridization Stringency Wash Buffer (2X) 375 ml Related Products Page 7701 Biodyne* B Nylon Membrane CL-XPosure Film North2South Biotin Random Prime DNA Labeling Kit Sufficient reagents for 10 reactions. Includes: 5X Heptanucleotide Mix 5X Deoxynucleotide Mix 10X Reaction Buffer Klenow DNA Polymerase Control DNA 5 M Ammonium Acetate 0.5 M EDTA, ph 8.0 Nuclease-free Water Biotin-11-UTP Kit 100 µl 80 µl 50 µl 10 µl 5 µl 1 ml 1 ml 1 ml 20 µl The Control DNA in this kit is covered under U.S. Patent No. 4,7,072. This Control DNA is intended for the research use described within the technical literature of the accompanying kit. No other use is authorized. Please contact Promega Corporation for applications relating to all such other cases. 194 To order or for technical support, contact your local office or distributor. See pages -2, 3.

69 Nucleic Acids Detection Reagents Chemiluminescent Nucleic Acid Detection Module A detection system for Northerns, Southerns, gel-shifts (EMSAs) and RPAs. The Thermo Scientific Chemiluminescent Nucleic Acid Detection Module is a complete system for detection of biotin-labeled nucleic acids for various blotting applications including Northern/Southern blots, ribonuclease protection assays (RPAs) and electrophoretic mobility shift assays (EMSAs). This module provides the essential detection components used in the Thermo Scientific North2South and LightShift EMSA Kits. The system includes an enhanced luminol substrate for horseradish peroxidase (HRP) with optimized blocking and wash buffers that together produce sensitivity equivalent to radioactive ( 32 P) systems. Obtain publication-quality blots having excellent signal and low background. Fast and sensitive achieve the same speed and detection levels you ve come to expect with the North2South and LightShift Kits Versatile kit protocol (included) is applicable to any biotin-based nucleic acid detection system: Northerns, Southerns, gel shifts, RPAs and colony lifts Flexible kit contains only the critical detection components for those who already have biotin-labeled probes and an optimized hybridization method developed Liver Brain Thymus Northern blot analysis of mouse total RNA. Total RNA samples (1 µg) prepared from the indicated tissues were electrophoresed on a denaturing 1% agarose gel, transferred to Biodyne* B Positive Nylon (Product # 7701), UV crosslinked and hybridized with a biotinylated c-myc/exon 3 RNA probe (4 ng/ml). Exposure time to film was 1 minute. D-Luciferin Heart Lung Spleen Purity and sensitivity at a reasonable price. 2.2 kb Thermo Scientific D-Luciferin is specially synthesized by our organic chemists to ensure high performance. We are the only supplier to perform chiral-high performance liquid chromatography (HPLC) testing to ensure that the product is not only pure but also in the active form. Our D-Luciferin outshines the competition on many levels. Testicle Ovary Kidney Embryo Chemiluminescent Nucleic Acid Detection Module Sufficient detection reagents for ten 10 x 10 cm membranes. Includes: Stabilized Streptavidin-Horseradish Peroxidase Conjugate Chemiluminescent Substrate Luminol/Enhancer Solution Stable Peroxide Solution Blocking Buffer 4X Wash Buffer Substrate Equilibration Buffer Kit 1.5 ml 80 ml 80 ml 500 ml 500 ml 500 ml Related Products Page # North2South Chemiluminescent Hybridization 194 Detection Kit North2South Complete Biotin Random 194 Prime Labeling and Detection Kit North2South Biotin Random Prime DNA 194 Labeling Kit LightShift Chemiluminescent EMSA Kit Biotin 3 End DNA Labeling Kit NE-PER Nuclear and Cytoplasmic Protein 11 Extraction Reagent CL-XPosure Film Biodyne B Nylon Membrane D-Luciferin, Monosodium Salt 5 mg 1515 D-Luciferin, Monosodium Salt 25 mg 1520 D-Luciferin, Monopotassium Salt 5 mg 1525 D-Luciferin, Monopotassium Salt 25 mg Additional dry ice and/or freight charges Additional hazardous handling charge *Trademark, see Trademark Index To order or for technical support, contact your local office or distributor. See pages -2,