Secondary Antibodies and Conjugates

Transcription

1 F(ab )2 Fragment Affinity-Purified Ordering Specialising in Secondary Antibodies and Conjugates Immunofluorescence Western Blotting Flow Cytometry In Situ Hybridization Electron Microscopy Enzyme Immunohistochemistry STED Microscopy 2-photon Microscopy ELISA

2 Contact and Ordering Information Who We Are Jackson ImmunoResearch Laboratories, Inc. specialises in the production and conjugation of affinity-purified secondary antibodies and purified immunoglobulins. We have strict quality control standards giving our products an unparalleled reputation among the scientific community for reliability and reproducibility. Our range includes Affinity Purified Whole IgG, F(ab') 2 Fragment and Monovalent Fab Fragment Antibodies with a multitude of species and conjugate options. There are in addition to our Flow Cytometry and Western Blot optimised antibodies, an extensive range of blocking reagents and much more, providing a comprehensive solution for immunological applications. Our goal is to provide research scientists with the largest selection of high quality secondary reagents and the best technical and customer services possible. From our European hub we offer Euro currency trading with fast delivery. All Jackson ImmunoResearch operations, including the sales, technical and distribution services of Jackson ImmunoResearch Europe comply with industry standards and are accredited with ISO 9001:2008 certification. Ordering Information For further information about any of our products and services please call us anytime during normal office hours (GMT), which are: hrs to 17.00hrs Monday to Thursday 08.30hrs to 16.00hrs Friday We accept orders by any of the following methods: Telephone: +44 (0) Fax: +44 (0) cuserv@jireurope.com Address: Unit 7 Acorn Business Centre Oaks Drive Newmarket CB8 7SY UK Please provide the following information: Delivery and Invoice Address VAT Number (if applicable) Product Code(s) required You can also order via our online shopping cart at Conditions: Technical Assistance ALL PRODUCTS LISTED IN THIS CATALOGUE ARE FOR IN VITRO RESEARCH USE ONLY. No product listed in this catalogue is a medical device. They are not intended for diagnostic or therapeutic purposes. Nothing disclosed herein is to be construed as a recommendation to use our products in violation of any patents. Jackson ImmunoResearch cannot be held responsible for patent infringements or other violations that may occur with the use of these products. Jackson ImmunoResearch makes no implied or other warranty of merchantability or fitness for a particular use and in no event shall Jackson ImmunoResearch be responsible for any consequential or other damages beyond the cost of replacing any product of Jackson ImmunoResearch. Customer agrees to indemnify and hold Jackson ImmunoResearch harmless and to reimburse Jackson ImmunoResearch for all costs related to any claim arising as a result of customer's use of any Jackson ImmunoResearch product contrary to the terms contained herein or otherwise specified in other documentation. Our highly qualified and dedicated team of scientists are ready to assist you. tech@jireurope.com and we will be delighted to help. 2

3 Contents Ordering Technical Ordering Information...inside front cover Technical Information - Affinity-Purified Secondary Antibodies...4 Selection and Location of Affinity-Purified Secondary Antibodies...4 Multiple Labelling (ML) Using Conjugated Secondary Antibodies...7 Probes Conjugated to Affinity-Purified Antibodies and to Other Proteins...8 Fluorescent Dyes...8 Alexa Fluor Cyanine (Cy TM 2, Cy3, Cy5) DyLight Aminomethylcoumarin Acetate (AMCA)...10 Fluorescein Isothiocyanate (FITC/DTAF)...11 Rhodamine Red TM -X (RRX)...12 Biotin-SP TM (long spacer)...13 Enzymes...13 Whole IgG Affinity-Purified Secondary Antibodies F(ab') 2 Fragment Affinity-Purified Secondary Antibodies Monovalent Fab Fragment Affinity-Purified Secondary Antibodies for Blocking and for Double Labelling Primary Antibodies from the Same Host Species Secondary Antibodies for Flow Cytometry IgG Controls and Streptavidin Conjugates for Flow Cytometry...58 Cy2, Cy3 and Cy5 Conjugates for Permanent Mounting...68 Streptavidin...71 ImmunoGold Reagents...72 LM Grade 4 nm Colloidal Gold - Antibody Complexes...73 EM Grade 6 nm, 12 nm & 18 nm Colloidal Gold - Antibody Complexes...74 Peroxidase-Anti-Peroxidase (PAP) Soluble Immune Complexes...75 Solid-Phase Immunoadsorbent Gels...75 Primary Antibodies for Signal Enhancement...76 Purified IgG Fraction Monoclonal Mouse Anti-Digoxin...76 Purified IgG Fraction Monoclonal Mouse Anti-Biotin...76 Purified IgG Fraction Monoclonal Mouse Anti-Fluorescein...76 Affinity-Purified Anti-Horseradish Peroxidase...76 Normal Serums and Gamma Globulins...78 Bovine Serum Albumin (IgG-Free, Protease-Free)...79 Purified Proteins from Normal Serums Antisera to Immunoglobulins, Whole Serums and Enzymes...90 Trademarks and Licences... inside back cover Anti-Mouse IgG Subclass Specific Antibodies...60 Anti-IgG, Light Chain Specific Antibodies for Western Blotting after Immunoprecipitation (IP)...62 Alexa Fluor 680 and Alexa Fluor 790 Conjugates for High Sensitivity Western Blots Telephone: +44 (0) Fax: +44 (0)

4 Technical Information - Affinity-Purified Secondary Antibodies Affinity-purified antibodies are isolated from antisera by immunoaffinity chromatography using antigens coupled to agarose beads. A proprietary elution process is used to dissociate antibodies from the antigen. Unconjugated affinity-purified antibodies are supplied sterile-filtered in phosphate buffer without stabilisers or preservatives. Conjugated affinity-purified antibodies are freeze-dried in phosphate buffer with stabilisers and sodium azide, with the exception of horseradish peroxidase conjugates, which do not contain a preservative. Alkaline phosphatase conjugates are freeze-dried in Tris buffer with stabilisers and sodium azide. L H lgg H L pepsin digestion L H F(ab ) 2 + fragments H L over digestion Fab H L Selection and Location of Affinity-Purified Secondary Antibodies Step 1. Affinity-purified secondary antibodies are offered in three different forms. Select from Whole IgG (pages 14-35), F(ab') 2 fragment (pages 36-45), or Fab fragment (pages 46-52) antibodies. Whole IgG (pages 14-35) antibodies are isolated as intact molecules from antisera by immunoaffinity chromatography. They have an Fc portion and two antigen binding Fab portions joined together by disulphide bonds (Figure 1) and therefore they are divalent. The average molecular weight is reported to be about 160 kda. The whole IgG form of antibodies is suitable for the majority of immunodetection procedures and is the most cost effective. F(ab') 2 fragment (pages 36-45) antibodies are generated by pepsin digestion of whole IgG antibodies to remove most of the Fc region while leaving intact some of the hinge region. F(ab') 2 fragments have two antigen-binding Fab portions linked together by disulphide bonds and therefore they are divalent. The average molecular weight is about 110 kda. They are used for specific applications, such as to avoid binding of secondary antibodies to live cells with Fc receptors or to Protein A or Protein G. However, binding of primary antibodies to Fc receptors also may occur if they are whole IgG antibodies, creating background regardless of the form of the secondary antibody. To block whole IgG primary and secondary antibodies from binding to Fc receptors, incubate L H lgg H L papain digestion H Fab H L cells in buffer containing 5% normal serum from the host species of the labelled secondary antibody. To prevent capping, endocytosis and regeneration of Fc receptors on living cells, incubate at 4 C in buffer containing 5% normal serum with sodium azide added to inhibit metabolism. Caution: Never block with normal serum or IgG from the host species of the primary antibody. If immunoglobulins in normal serum bind to the specimen of interest, they will be recognised by the labelled secondary antibody, resulting in higher background. Bovine serum albumin (BSA) and dry milk, both commonly used for blocking, may contain bovine IgG. With the exception of Bovine Anti-Goat IgG, many secondary antibodies such as Anti-Bovine, Anti-Goat and Anti-Sheep will react strongly with bovine IgG. Therefore, use of BSA or dry milk for blocking or diluting these antibodies may significantly increase background and/or reduce antibody titre. For blocking, use normal serum (5% v/v) from the host species of the labelled secondary antibody. + H H Figure 1. Schematic representation of IgG fragments generated by enzymatic digestions. Figure 1. Fc 4

5 Ordering Technical Technical Information - Affinity-Purified Secondary Antibodies Fab fragment (pages 46-52) antibodies are generated by papain digestion of whole IgG antibodies to remove the entire Fc portion, including the hinge region (Figure 1). These antibodies are monovalent, containing only a single antigen binding site. The molecular weight of Fab fragments is about 50 kda. They can be used to block endogenous immunoglobulins on cells, tissues, or other surfaces and to block the exposed immunoglobulins in multiple labelling experiments using primary antibodies from the same species. In contrast, divalent (whole IgG or F(ab') 2 fragment) antibodies should not be used for blocking since they have two binding sites. After blocking, some of the binding sites would be available to capture the primary antibody introduced in a subsequent step, resulting in higher background and/or coincidental labelling. Step 2. Select the secondary antibody. The antibodies are listed alphabetically according to the host species of the primary antibody. For example, if the primary antibody is made in mouse, go to the "Anti-Mouse" section. Note: Both anti-syrian and anti-armenian hamster secondary antibodies are listed under "Anti-Hamster". It is important to know in which strain of hamster the primary antibody was produced since cross-reaction between the strains is not complete. Step 3. Select the host species of the secondary antibody. Selection of the host species for a secondary antibody involves many considerations, including but not limited to: 1) Antibodies from some host species may not be adsorbed against cross-reacting species of interest. Choose a host species with the required adsorptions. 2) Host species compatibility. Some host species may not be compatible with other species in multiple-labelling experiments. In general, all secondary antibodies for multiple labelling should come from the same host species. 3) Binding to Protein A and Protein G. Rabbit antibodies bind well to Protein A and Protein G, but goat and donkey antibodies bind better to Protein G. 4) Personal preference or experience. In our experience there appears to be no host species-specific difference in the quality of secondary antibodies. Step 4. Select the secondary antibody specificity under "Antibody Description". The following explanations of terms may assist in selecting the most appropriate antibody specificity. Note: Immunoglobulins from different species share similar structures, with similarities being related to closeness in phylogeny. Antibodies against immunoglobulins from one species may cross-react with a number of other species unless they have been specifically adsorbed against the cross-reacting species. Antibodies that have been adsorbed against other species will contain "(min X...Sr Prot)" in the antibody description. Anti-IgG (H+L) These antibodies react with both the heavy and light chains of the IgG molecule, i.e. with both the Fc and F(ab') 2/Fab portions of IgG (Figure 1). Anti-IgG (H+L) antibodies also react with other immunoglobulin classes (IgM, IgA, IgD, IgE) and subclasses since they all share the same light chains (either kappa or lambda). Anti-IgG (H+L) antibodies have broader epitope recognition than anti-fragment specific antibodies. They are suggested for all general immunodetection procedures. Anti-IgG, Fc/Fc γ fragment specific These antibodies react with the Fc portion of the IgG heavy chain. They have been tested by ELISA and/or adsorbed against Fab fragments. In some cases, they are additionally tested and/or adsorbed to minimise cross-reactivity to IgM and/or IgA. In such cases (antihuman, anti-mouse and anti-rat), they are labelled "Anti-IgG, Fc γ". Caution: Anti-IgG, Fc γ fragment specific antibodies may not react equally with all monoclonal primary antibodies. For an anti-mouse IgG, Fc γ fragment specific antibody with balanced reactivity to four subclasses of IgG, select goat anti-mouse IgG (subclasses 1+2a+2b+3), Fc γ fragment specific (min X Hu, Bov, Rb Sr Prot). Anti-Mouse IgG, Fc γ Subclass specific These antibodies react with the Fc portion of the heavy chain of individual subclasses of mouse IgG. They have been tested by ELISA and adsorbed to minimise cross-reactivity to Telephone: +44 (0) Fax: +44 (0)

6 Technical Information - Affinity-Purified Secondary Antibodies other subclasses, Fab fragments, IgM and a few other species of IgG. Anti-Mouse IgG, Fc γ subclass specific antibodies react with individual subclasses of mouse IgG. They are intended for distinguishing between different subclasses of mouse IgG primary antibodies in multiple labelling experiments, or for IgG subclass determination. Anti-IgG, F(ab') 2 fragment specific These antibodies react with the F(ab') 2/Fab portion of IgG. They have been tested by ELISA and/or adsorbed against Fc fragments. They are not specific for IgG since they react with light chains and therefore also react with other immunoglobulin classes (IgA, IgM, IgD and IgE) and subclasses sharing the same light chains. (min X... Sr Prot) Secondary antibodies against one species may cross-react with other species unless they have been specifically adsorbed against the other species. Antibodies with "(min X... Sr Prot)" in the description have been tested and/or adsorbed against IgG and/or serum proteins of those species indicated in the parentheses. They are recommended when the presence of immunoglobulins from other species may lead to interfering cross-reactivities. However, caution should be exercised when considering antibodies that have been adsorbed against closely related species, since they have greatly reduced epitope recognition and may recognise some monoclonals poorly. For example, only use antimouse IgG adsorbed against rat IgG to detect a mouse primary antibody in rat tissue which contains endogenous rat immunoglobulins, or in a multiple labelling application which includes a rat primary antibody. Use anti-mouse IgG not adsorbed against rat IgG to detect a mouse primary antibody in the absence of rat immunoglobulins. Two other examples of antibodies which have diminished epitope recognition after adsorption with closely related species are Anti-Rat IgG (min X... Mouse... Sr Prot) and Anti-Armenian Hamster IgG (min X... Mouse, Rat... Sr Prot). Refer to "ML (Multiple Labelling)" for further information. The following abbreviations are used in the parentheses: min X = minimal cross-reaction Bov = Bovine Ck = Chicken Gt = Goat GP = Guinea Pig Ar Hms = Armenian Hamster Sy Hms = Syrian Hamster Hrs = Horse Hu = Human Ms = Mouse Rb = Rabbit Shp = Sheep Sw = Swine Sr = Serum Prot = Protein ML (multiple labelling) Some antibodies are designated "ML" to emphasise their usefulness in multiple labelling in addition to single labelling. For further information see: "Multiple Labelling (ML) Using Labelled Secondary Antibodies" (page 7). Anti-Armenian Hamster IgG vs Anti-Syrian Hamster IgG Most hamster monoclonal antibodies are derived from Armenian hamster spleen cell-mouse myeloma hybridomas. The IgG produced by these hybridomas is Armenian (not Syrian) hamster IgG. Most commercially available polyclonal anti-hamster IgG antibodies have been anti-syrian hamster IgG, which are not as effective as anti-armenian hamster IgG in detecting Armenian hamster IgG monoclonal antibodies. Caution: Anti-Armenian Hamster IgG (H+L) (min X Bov, Hu, Ms, Rb, Rat Sr Prot) may not recognise all Armenian hamster monoclonal antibodies, since it has been adsorbed against closely related species (in bold). Therefore, it is better to use an antibody adsorbed against fewer species, such as Anti-Armenian Hamster IgG (H+L) (min X Bov Sr Prot), except in those cases where Armenian hamster monoclonals need to be detected in the presence of mouse and/or rat immunoglobulins. Step 5. Select the desired probe from those listed at the top of the product tables. For technical information about probes, see "Technical Information on Probes Conjugated to Affinity-Purified Antibodies and to Other Proteins" (page 8). 6

7 Technical Information - Affinity-Purified Secondary Antibodies Ordering cuserv@jireurope.com Technical tech@jireurope.com Step 6. Find the size and code number of the selected antibody by following the row until it intersects the column of the desired probe. The top numbers in each cell refer to the unit size. The bottom nine-digit number is the catalogue code number. Step 7. Complete product description for ordering purposes. For a complete description of the product, please use the standard format to avoid mistakes when placing an order. For example, a product with the code number should be described as in the key below. FITC-conjugated AffiniPure F(ab')2 fragment Goat Anti-Mouse IgG, F(ab')2 fragment specific (min X Hu, Bov, Hrs Sr Prot) A B C D E F G A. Description of the probe, if it is conjugated. If unconjugated, nothing is required here. B. AffiniPure is our trade name for antibodies which have been isolated from antisera by immunoaffinity chromatography using antigens coupled to agarose beads. C. Form of the antibody - whole IgG, F(ab')2 fragment, or Fab fragment antibody. D. Name of the host species of the secondary antibody. E. Name of the species with which the antibody reacts. F. Description of the antibody specificity. G. List of species against which the antibody has been adsorbed to minimise cross-reactivity. Multiple Labelling (ML) Using Labelled Secondary Antibodies Selection of antibodies for simultaneous detection of more than one antigen depends on at least two important criteria: 1. Availability of secondary antibodies that do not recognise (a) one another (are derived from the same host species), (b) other primary antibodies used in the assay system, (c) immunoglobulins from other species present in the assay system, or (d) endogenous immunoglobulins present in the tissues or cells under investigation. 2. Use of probes (enzyme-reaction products, fluorophores, or electron-dense particles) that are well resolved. The affinity-purified antibodies marked "ML" (multiple labelling) have been specifically prepared to meet these criteria. One of many possible multiple-labelling protocols using these reagents is shown in the following example. Mouse Tissue Antigen A Mouse Tissue Antigen B Mouse Tissue Antigen C Step 1: 5% N. Donkey Serum to block Step 2: Goat Anti-Antigen A Step 4: 5% N. Donkey Serum to block (if needed) Step 5: Rabbit Anti-Antigen B Step 7: 5% N. Donkey Serum to block (if needed) Step 8: Rat Anti-Antigen C Key to the format of product descriptions eg: FITC-conjugated AffiniPure F(ab') 2 fragment Goat Anti-Mouse IgG, F(ab') 2 fragment specific (min X Hu, Bov, Hrs, Sr Prot) Step 3: Probe l-conjugated Donkey Anti-Goat IgG (H+L) (min X Ck, GP, Sy Hms, Hrs, Hu, Ms, Rb, Rat Sr Prot) Step 6: Probe ll-conjugated Donkey Anti-Rabbit IgG (H+L) (min X Bov, Ck, Gt, GP, Sy Hms, Hrs, Hu, Ms, Rat, Shp Sr Prot) Step 9: Probe lll-conjugated Donkey Anti-Rat IgG (H+L) (min X Bov, Ck, Gt, GP, Sy Hms, Hrs, Hu, Ms, Rb, Shp Sr Prot) Note: Wash thoroughly after each step, including after blocking at step 1. With heavy or persistent background further blocking may be required at Steps 4 and 7. Do not dilute any antibody with normal serum or mix antibodies together to save time, which may result in immune complex formation which could increase background. In this example, the secondary antibodies used in Steps 3, 6 and 9 do not recognise each other since they are all made in donkey. They have been solid-phase adsorbed so that they do not recognise the other primary antibodies used in Steps 2, 5 and 8. Also, they do not react with endogenous mouse Ig, which may be present in the mouse tissue. For a review of multi-colour immunofluorescence labelling with confocal microscopy see Brelje, Wessendorf and Sorenson, "Multi-colour laser scanning confocal immunofluorescence microscopy: Practical application and limitations." In Cell Biological Applications of Confocal Microscopy (Methods in Cell Biology. vol. 38). Ed. B. Matsumoto. Orlando, FL: Academic Press, Inc. 1993, pp Telephone: +44 (0) Fax: +44 (0)

8 Technical Information - Probes Technical Information on Probes Conjugated to Affinity-Purified Antibodies and to Other Proteins. Fluorescent Dyes (for Phycoerythrin, PerCP and Allophycocyanin conjugates, see page 53). The selection of fluorophores depends on: A. Instrument set-up. Examples include availability of light sources, filter sets and detection systems. B. Degree of colour separation desired for multiple labelling. For example, to achieve good colour separation from Alexa Fluor 488, choose a shorter wavelength emitting fluorophore such as DyLight 405 and/or longer wavelength-emitting fluorophores such as Rhodamine Red-X, Alexa Fluor 594, or Alexa Fluor 647. C. Sensitivity required. For example, Alexa Fluor 488 is brighter than FITC. The following fifteen fluorescent dye conjugates (Figure 2, Table 1) are currently available from Jackson ImmunoResearch. They cover the most commonly used excitation sources and filter sets from blue to infrared emissions. Alexa Fluor Fluorescent Dyes Alexa Fluor fluorescent dyes are widely recognised as superior fluorescent dyes available for conjugation. They are highly water soluble and remain fluorescent from ph 4 to ph 10. The detection level of any fluorophore-antibody conjugate depends on brightness and photostability of the dye; antibody activity, specificity and cross-reactivity; and the optimal moles of dye per mole of antibody. These parameters have been researched for each dye conjugate to optimise the level of antibody detection and minimise background. Cyanine dyes (Cy2, Cy3 and Cy5) Among currently available fluorescent dyes, the cyanine dyes are better able to withstand the harsh dehydration and embedding conditions required for mounting sections in nonpolar plastic mounting media, such as DPX and Permount. The cyanine dyes are Fluorophore DyLight 405 Aminomethylcoumarin, AMCA Cyanine, Cy2 Alexa Fluor 488 Fluorescein, FITC/DTAF Indocarbocyanine, Cy3 R-Phycoerythrin R-PE Rhodamine Red-X, RRX Alexa Fluor 594 Allophycocyanin APC Alexa Fluor 647 Indodicarbocyanine, Cy5 Peridinan-Chlorophyll-protein, PerCP Alexa Fluor 680 Alexa Fluor 790 Excitation Peak many, many, many, Emission Peak (nm) Table 1. Approximate peak wavelengths of excitation and emission for all fluorophore-conjugated, affinity-purified secondary antibodies offered by Jackson ImmunoResearch in order by increasing peak of emission. Approximate values are given for purposes of comparing one fluorophore with another. Actual values may vary depending on the spectrofluorometer used in each laboratory. brighter in the non-polar environment than in an aqueous medium, resulting in less acquisition time than other dyes in the confocal microscope. The advantage of using plastic mounting media is better long-term storage for re-examination and retrieval of data at a later date. See pages for a list of cyanine dye conjugates. 8

9 Technical Information - Probes Ordering cuserv@jireurope.com Technical tech@jireurope.com Excitation Fluorescence Figure 2. Excitation (left) and emission (right) spectra of all fluorophores offered by Jackson ImmunoResearch. The conjugates in order of increasing wavelength of fluorescence are DyLight 405 (violet), AMCA (dark blue), Cy2 (light blue), Alexa Fluor 488 (blue-green), FITC (hidden behind Alexa Fluor 488), Cy3 (yellow), R-PE (dark pink, dotted), Rhodamine Red-X (orange), Alexa Fluor 594 (red), Allophycocyanin (pink, dotted), Cy5 (green), Alexa Fluor 647 (purple-hidden behind Cy5), PerCP (lavender, dotted), Alexa Fluor 680 (grey) and Alexa Fluor 790 (black) Wavelength (nm) Wavelength (nm) 850 This figure illustrates the relative shape and position of each fluorophore in the peak region of its excitation and fluorescence emission following conjugation to antibodies. Quantitative comparisons should not be made since peak heights have been normalised. All spectra were obtained with an M-Series spectrofluorometer system from Photon Technology International, Inc. Figure 2. Figure 3. Excitation (left) and Emission (right) spectra of Alexa Fluor conjugated affinity-purified secondary antibodies, streptavidin and purified proteins. The dyes are Alexa Fluor 488 (blue), Alexa Fluor 594 (green), Alexa Fluor 647 (red), Alexa Fluor 680 (pink) and Alexa Fluor 790 (black). Excitation Fluorescence This figure illustrates the relative shape and position of each fluorophore conjugate in the peak region of its excitation and fluorescence emission. Quantitative comparisons should not be made since peak heights have been normalised. All spectra were obtained with an M-Series spectrofluorometer system from Photon Technology International, Inc Wavelength (nm) Wavelength (nm) 850 Figure 3. Telephone: +44 (0) Fax: +44 (0)

10 Technical Information - Probes Figure 4. Excitation (left) and emission (right) spectra of Cy2 (green), Cy3 (red) and Cy5 (purple). Peak heights were normalised after the spectra were obtained with an M-series spectrofluorometer system from Photon Technology International, Inc. Excitation Wavelength (nm) Figure 4. Fluorescent Dyes in Order of Increasing Emission Wavelength A brief description of the characteristics of fluorophores found in this catalogue is found below. The fluorophores are listed in order of increasing emission wavelength (Figure 2 and Table 1). For Phycoerythrin, PerCP and Allophycocyanin see page 53. DyLight 405-conjugated secondary antibodies are excited maximally at about 400 nm and fluoresce with a peak at about 421 nm (Figure 2 and Table 1). They are very bright and photostable, but their optimal use is limited to confocal microscopes equipped with a 405 nm laser and a 420 nm emission filter. Under these conditions, it is possible to perform effective 4-colour imaging with good colour separation, good photostability and high sensitivity. The combination of DyLight 405, Alexa Fluor 488, Rhodamine Red-X and Alexa Fluor 647 provides for maximum colour separation (Figure 5). Another 4-colour dye combination, which may be equally effective but has slightly less colour separation, is DyLight 405, Alexa Fluor 488, Cy3 and Alexa Fluor 647. DyLight 405 is not recommended for use in epifluorescence microscopes, nor is it recommended for flow cytometry, because emission filters generally used in flow cytometers are not optimal for DyLight 405. DyLight 405 conjugates are the best choice for blue-fluorescing secondary antibodies in multi-colour labelling protocols. See tables of Whole IgG Affinity-Purified Antibodies (pages 14-35), Streptavidin (page 71) and Purified Proteins from Normal Serums (pages 80-89) for all DyLight 405 conjugates. AMCA (Aminomethylcoumarin Acetate) conjugates absorb light maximally around 350 nm and fluoresce maximally around 450 nm (Figure 2 and Table 1). For fluorescence microscopy, AMCA can be excited with a mercury lamp and observed using a UV filter set. Since blue fluorescence is not well detected by the human eye, AMCA-conjugated secondary antibodies should be used only with the most abundant antigens in multiplelabelling experiments. Ways of improving the visibility of AMCA include dark adapting the eyes, using fluorite instead of glass objectives, avoiding mounting media that absorb UV light (such as plastic-based media) and capturing photographic images with blue-sensitive film or CCD cameras. AMCA fades rapidly in conventional epifluorescence and confocal 10

11 Technical Information - Probes Ordering cuserv@jireurope.com Technical tech@jireurope.com microscopy and therefore it should be used with mounting media containing an anti-fading agent such as n-propyl gallate. For flow cytometry, AMCA can be excited with a mercury lamp or with a water-cooled argon ion laser which emits some lines in the UV. AMCA has been used mostly for multiple labelling since there is minimal fluorescence overlap with green-fluorescing dyes and little or no overlap with longer wavelength-emitting fluorophores. Applications for multiple labelling with this probe include both immunofluorescence microscopy and flow cytometry. AMCA is not suggested for single labelling in one-photon microscopy because of its relatively weak signal and rapid fading. However, AMCA has been found to be a bright and photostable dye for 2-photon microscopy. In one-photon microscopy DyLight 405 is a better choice for multiple labelling. Cy2 conjugates have maximum absorption/excitation at 492 nm and fluoresce with a peak around 510 nm in the green region of the visible spectrum (Figure 2 and Table 1) like FITC conjugates (520 nm), but they are more photostable and less sensitive to ph changes than FITC. However, for mounting in aqueous media we recommend Alexa Fluor 488 as the preferred green-fluorescing dye because it is brighter and more photostable than Cy2, FITC and DyLight 488. A further disadvantage of using Cy2 with aqueous mounting media is its sensitivity to p-phenylenediamine, an anti-fading agent found in some commercial mounting media, which results in weak and diffused fluorescence after storage of stained slides. The main advantage of Cy2 conjugates is increased fluorescence in plastic mounting media. Cy2 is available conjugated to a limited selection of antibodies (pages 68-70). Alexa Fluor 488-conjugated antibodies absorb light maximally at 493 nm and fluoresce with a peak around 519 nm (Figure 2 and Table 1). In aqueous mounting media they are brighter than FITC, Cy2 and DyLight 488. Alexa Fluor 488 conjugates are recommended for maximum sensitivity for all immunofluorescence procedures requiring a green-fluorescing dye, except for protocols that include mounting in plastic mounting media. FITC (Fluorescein isothiocyanate) is the form of fluorescein used for conjugation to all of our antibodies and purified proteins, with the exception of streptavidin. Fluorescein conjugates absorb light maximally at 492 nm and fluoresce maximally at 520 nm (Figure 2 and Table 1). Although less bright than other green-fluorescing dyes, FITC is still a widely used fluorophore due to its long history. The major disadvantage of fluorescein is its rapid photobleaching (fading), which can be mitigated by the use of an anti-fading agent in the mounting medium. A better choice for many applications involving FITC is Alexa Fluor 488 because it is brighter and more photostable. DTAF (Dichlorotriazinylamino fluorescein) is another form of fluorescein, with excitation and emission peaks identical to those of FITC. We use DTAF (instead of FITC) only for conjugation with streptavidin, since fluorescence from FITC is greatly quenched after conjugation with streptavidin. This phenomenon is unique to streptavidin and is not observed with antibodies. Cy3 is brighter, more photostable and gives less background than other orange-red fluorescing dye conjugates. Cy3 conjugates can be excited maximally at 550 nm, with peak emission at 570 nm (Figure 2 and Table 1). For fluorescence microscopy, Cy3 can be visualised with traditional tetramethyl rhodamine (TRITC) filter sets, since the excitation and emission spectra are nearly identical to those of TRITC. We recommend Cy3 as a brighter alternative to TRITC. Cy3 can be excited to about 50% of maximum with an argon laser (514 nm or 528 nm lines), or to about 75% of maximum with a helium/neon laser (543 nm line) or mercury lamp (546 nm line). Cy3 has been used with fluorescein for double labelling; however, the use of a narrow band-pass emission filter for fluorescein is recommended to minimise Cy3 fluorescence in the FITC filter set. Cy3 can also be paired with Alexa Fluor 647 for multiple labelling when using a confocal microscope. However, a better choice for multiple labelling is Rhodamine Red-X (Figure 2 and Table 1) because its fluorescence is midway between a green fluorescing dye (like Alexa Fluor 488) and a farred-fluorescing dye like Alexa Fluor 647 (Figure 5). RRX (Rhodamine Red-X) conjugates have a peak of excitation at 570 nm and a peak of emission at 590 nm (Figure 2 and Table 1). Although TRITC has been used traditionally with FITC for double labelling, better colour separation is achieved by using RRX or Alexa Fluor 594. Rhodamine Red-X is particularly useful for 3- and 4-colour labelling with Telephone: +44 (0) Fax: +44 (0)

12 Technical Information - Probes DyLight 405, Alexa Fluor 488 and Alexa Fluor 647 by using a confocal microscope equipped with a 405 nm laser and a krypton/argon laser. Fluorescence from RRX lies about midway between that of Alexa Fluor 488 and Alexa Fluor 647 and it shows little overlap with either dye (Figure 5). The krypton-argon laser emits lines at 488 nm, 568 nm and 647 nm, which are optimal for exciting Alexa Fluor 488, RRX and Alexa Fluor 649, respectively. By adding a 405 nm laser and a 420 nm emission filter, 4-colour labelling is possible using DyLight 405-conjugated secondary antibodies from JIR (Figure 5). The separation between all four dyes is perfect for 4-colour labelling and all four dyes are very bright. Fluorescence Figure 5. Emission spectra for optimal 4-colour imaging using DyLight 405 (blue), Alexa Fluor 488 (green), Rhodamine Red-X (red) and Alexa Fluor 647 (purple). Alexa Fluor 594-conjugated antibodies absorb light maximally around 591nm and fluoresce with a peak around 614 nm (Figure 2 and Table 1). They are brighter, more photostable and more hydrophilic than Texas Red conjugates. Alexa Fluor 594 conjugates are brighter than red-fluorescing conjugates and they provide more colour separation from green-fluorescing dyes than DyLight 549, Cy3 and TRITC conjugates. They are the best choice for immunofluorescence detection in the deep-red region of the visible spectrum. Figure Wavelength (nm) Alexa Fluor 647-conjugated antibodies absorb light maximally around 651 nm and fluoresce maximally around 667 nm (Figures 2 and 3 and Table 1). They are brighter than Cy5 and DyLight 650 in aqueous mounting media. Alexa Fluor 647- and APCconjugated secondary antibodies are the best choice for flow cytometry when secondary antibodies fluorescing at these wavelengths are desired. Alexa Fluor 647 conjugates are the best choice of far red-emitting dyes for multiple-labelling detection with a confocal microscope. A significant advantage of using Alexa Fluor 647 over lower wavelength-emitting dyes is the low autofluorescence of biological specimens in this region of the spectrum. However, because of its peak emission at 667 nm, Alexa Fluor 647 cannot be seen well by eye and it cannot be excited optimally with a mercury lamp. Therefore, Alexa Fluor 647 is not recommended for use with conventional epifluorescent microscopes. It is most commonly visualised with a confocal microscope equipped with an appropriate laser for excitation and a far-red detector. Alexa Fluor 647 conjugates are less expensive alternatives to allophycocyanin conjugates for flow cytometry. Cy5 conjugates are excited maximally at 650 nm and fluoresce maximally at 670 nm (Figure 2 and Table 1). They can be excited to about 98% of maximum with a krypton/argon laser (647 nm line) or to about 63% of maximum with a helium/neon laser (633 nm line). Cy5 can be used with a variety of other fluorophores for multiple labelling due to a wide separation of its emission from that of shorter wavelength-emitting fluorophores. However, we recommend Alexa Fluor 647 as the preferred far-red-fluorescing dye because it is brighter than Cy5 in aqueous mounting media. The reverse is true when mounting in nonpolar, plastic mounting media, i.e. Cy5 is brighter than other far-red-fluorescing dyes, including Alexa Fluor 647 and DyLight 650. Cy5 is available conjugated to a limited selection of antibodies, see pages Alexa Fluor 680 and Alexa Fluor 790 conjugates are used for very sensitive Western blots, ELISAs and multiplexing arrays. Alexa Fluor 680 conjugates are excited with a peak around 684 nm and fluoresce with a peak around 702 nm (Figures 2 and 3 and Table 1). Alexa Fluor 790 conjugates are excited with a peak around 792 nm and 12

13 Technical Information - Probes Ordering cuserv@jireurope.com Technical tech@jireurope.com fluoresce at a peak around 803 nm. They are the best choice for highly sensitive single or double labelling with fluorescence imaged in a LiCor Odyssey imager (see page 64 for more information). Biotin-SP (long spacer) Biotin-SP is our trade name for biotin with a 6-atom spacer positioned between biotin and the protein to which it is conjugated. When Biotin-SP-conjugated antibodies are used in enzyme immunoassays, there is an increase in sensitivity compared to biotin-conjugated antibodies without the spacer. This is especially notable when Biotin-SP conjugated antibodies are used with alkaline phosphatase-conjugated streptavidin. Apparently, the long spacer extends the biotin moiety away from the antibody surface, making it more accessible to binding sites on streptavidin. Biotinylated antibodies require an additional reagent for visualisation. We offer streptavidin (page 71) and Mouse Anti-Biotin conjugated to fluorophores and enzymes (pages 76-77). Alkaline phosphatase (from calf intestine) conjugates are prepared by a modified method of Avremeas et al., Scand. J. Immunol (Supple. 7), 7. Resulting conjugates contain heterogeneous, high molecular weight complexes. They are sensitive reagents for solid-phase immunoassays such as ELISA and Western blotting. Although alkaline phosphatase conjugates are sometimes used for immunohistochemistry, penetration into whole mount tissues may be limited by their large sizes. Enzymes Horseradish peroxidase (HRP) conjugates are prepared by a modified Nakane and Kawaoi procedure (J. Histochem. Cytochem , 1084). Peroxidase conjugates are commonly used for immunohistochemistry, Western blotting and ELISA. Affinitypurified anti-horseradish peroxidase and conjugates are available for detection of horseradish peroxidase antigen or for signal amplification of HRP-containing reagents. For immunostaining of mammalian cells, an advantage of using anti-horseradish peroxidase is reduced background, because the antibody does not recognise the endogenous peroxidase-like enzymes found in those cells. Telephone: +44 (0) Fax: +44 (0)

14 Whole IgG Affinity-Purified Antibodies Antibody Description Unconjugated DyLight 405 A=400, E=421 Coumarin AMCA A=350, E=450 Alexa Fluor 488 A=493, E=519 Whole IgG ANTI-BOVINE Goat Anti-Bovine IgG (H+L)*** Goat Anti-Bovine IgG (H+L)*** (min X Ar Hms, Hu, Ms, Rat Sr Prot) Rabbit Anti-Bovine IgG (H+L)*** Whole IgG ANTI-CAT Goat Anti-Cat IgG (H+L) Goat Anti-Cat IgG, Fc fragment specific Goat Anti-Cat IgG, F(ab') 2 fragment specific Whole IgG ANTI-CHICKEN Donkey Anti-Chicken IgY (IgG)(H+L) (min X Bov, Gt, GP, Sy Hms, Hrs, Hu, Ms, Rb, Rat, Shp Sr Prot) Goat Anti-Chicken IgY (IgG)(H+L) (min X Bov, Gt, GP, Sy Hms, Hrs, Hu, Ms, Rb, Rat, Shp Sr Prot) Rabbit Anti-Chicken IgY (IgG) (H+L) Rabbit Anti-Chicken IgY (IgG), Fc fragment specific Rabbit Anti-Chicken IgY (IgG), F(ab') 2 fragment specific Whole IgG ANTI-DOG Rabbit Anti-Dog IgG (H+L) *** Warning: BSA and dry milk may contain IgG which will be recognised by this antibody. Use of BSA or dry milk to block or dilute this antibody may increase background and/or reduce secondary antibody titre. * ML = Multiple Labelling (see Multiple Labelling on pages 6-7 for an explanation). IgY is the original designation for the IgG-like protein found in both serum and egg yolk.

15 Whole IgG Affinity-Purified Antibodies Ordering Technical Fluorescein FITC A=492, E=520 Cyanine Cy3 A=550, E=570 Rhodamine Red-X RRX A=570, E=590 Alexa Fluor 594 A=591, E=614 Alexa Fluor 647 A=651, E=667 Biotin-SP (long spacer) Horseradish Peroxidase Alkaline Phosphatase Telephone: +44 (0) Fax: +44 (0)

16 Whole IgG Affinity-Purified Antibodies Antibody Description Unconjugated DyLight 405 A=400, E=421 Coumarin AMCA A=350, E=450 Alexa Fluor 488 A=493, E=519 Whole IgG ANTI-DOG Rabbit Anti-Dog IgG, Fc fragment specific Whole IgG ANTI-GOAT Bovine Anti-Goat IgG (H+L) (min X Bov, Ck, GP, Sy Hms, Hrs, Hu, Ms, Rb, Rat Sr Prot) Donkey Anti-Goat IgG (H+L)*** Donkey Anti-Goat IgG (H+L)*** (min X Ck, GP, Sy Hms, Hrs, Hu, Ms, Rb, Rat Sr Prot) Mouse Anti-Goat IgG (H+L)*** (min X Hu, Ms, Rb Sr Prot) IgG Fraction Monoclonal Mouse Anti-Goat IgG, Light Chain Specific*** (min X Hrs, Hu, Ms, Rb, Rat Ig) Rabbit Anti-Goat IgG (H+L)*** Rabbit Anti-Goat IgG (H+L)*** (min X Hu Sr Prot) Rabbit Anti-Goat IgG, Fc fragment specific*** Rabbit Anti-Goat IgG, Fc fragment specific*** (min X Hu Sr Prot) Rabbit Anti-Goat IgG, F(ab') 2 fragment specific*** Rabbit Anti-Goat IgG, F(ab') 2 fragment specific*** (min X Hu Sr Prot) Whole IgG ANTI-GUINEA PIG Donkey Anti-Guinea Pig IgG (H+L) (min X Bov, Ck, Gt, Sy Hms, Hrs, Hu, Ms, Rb, Rat, Shp Sr Prot) *** Warning: BSA and dry milk may contain IgG which will be recognised by this antibody. Use of BSA or dry milk to block or dilute this antibody may increase background and/or reduce secondary antibody titre. * ML = Multiple Labelling (see Multiple Labelling on pages 6-7 for an explanation).

17 Whole IgG Affinity-Purified Antibodies Ordering Technical Fluorescein FITC A=492, E=520 Cyanine Cy3 A=550, E=570 Rhodamine Red-X RRX A=570, E=590 Alexa Fluor 594 A=591, E=614 Alexa Fluor 647 A=651, E=667 Biotin-SP (long spacer) Horseradish Peroxidase Alkaline Phosphatase Telephone: +44 (0) Fax: +44 (0)

18 Whole IgG Affinity-Purified Antibodies Antibody Description Unconjugated DyLight 405 A=400, E=421 Coumarin AMCA A=350, E=450 Alexa Fluor 488 A=493, E=519 Whole IgG ANTI-GUINEA PIG Goat Anti-Guinea Pig IgG (H+L) Goat Anti-Guinea Pig IgG, Fc fragment specific Goat Anti-Guinea Pig IgG, F(ab') 2 fragment specific Whole IgG ANTI-HAMSTER (Armenian Hamster) Goat Anti-Armenian Hamster IgG (H+L) (min X Bov Sr Prot) Goat Anti-Armenian Hamster IgG (H+L) (min X Bov, Hu, Ms, Rb, Rat Sr Prot)** Whole IgG ANTI-HAMSTER (Syrian Hamster) Goat Anti-Syrian Hamster IgG (H+L) (min X Bov, Hrs, Hu, Ms, Rb, Rat Sr Prot) Rabbit Anti-Syrian Hamster IgG (H+L) Whole IgG ANTI-HORSE Goat Anti-Horse IgG (H+L) Goat Anti-Horse IgG, Fc fragment specific Rabbit Anti-Horse IgG (H+L)*** Whole IgG ANTI-HUMAN Donkey Anti-Human IgG (H+L) (min X Bov, Ck, Gt, GP, Sy Hms, Hrs, Ms, Rb, Rat, Shp Sr Prot) *** Warning: BSA and dry milk may contain IgG which will be recognised by this antibody. Use of BSA or dry milk to block or dilute this antibody may increase background and/or reduce secondary antibody titre. ** Caution: See page 6 (min X... Sr Prot) before selecting an antibody adsorbed against closely related species. * ML = Multiple Labelling (see Multiple Labelling on pages 6-7 for an explanation).

19 Whole IgG Affinity-Purified Antibodies Ordering Technical Fluorescein FITC A=492, E=520 Cyanine Cy3 A=550, E=570 Rhodamine Red-X RRX A=570, E=590 Alexa Fluor 594 A=591, E=614 Alexa Fluor 647 A=651, E=667 Biotin-SP (long spacer) Horseradish Peroxidase Alkaline Phosphatase Telephone: +44 (0) Fax: +44 (0)

20 Whole IgG Affinity-Purified Antibodies Antibody Description Unconjugated DyLight 405 A=400, E=421 Coumarin AMCA A=350, E=450 Alexa Fluor 488 A=493, E=519 Whole IgG ANTI-HUMAN Donkey Anti-Human IgG, Fc γ fragment specific (min X Bov, Hrs, Ms Sr Prot) Donkey Anti-Human IgM, Fc 5µ fragment specific (min X Bov, Hrs Sr Prot) Goat Anti-Human IgG (H+L) Goat Anti-Human IgG (H+L) (min X Bov, Hrs, Ms Sr Prot) Goat Anti-Human IgG, Fc γ fragment specific Goat Anti-Human IgG, Fc γ fragment specific (min X Bov, Hrs, Ms Sr Prot) Goat Anti-Human IgG, F(ab') 2 fragment specific Goat Anti-Human IgG, F(ab') 2 fragment specific (min X Bov, Hrs, Ms Sr Prot) Goat Anti-Human IgG + IgM (H+L) Goat Anti-Human IgG + IgM (H+L) (min X Bov Sr Prot) Goat Anti-Human IgA + IgG + IgM (H+L) Goat Anti-Human IgM, Fc 5µ fragment specific Goat Anti-Human IgM, Fc 5µ fragment specific (min X Bov Sr Prot) Goat Anti-Human Serum IgA, α chain specific Mouse Anti-Human IgG (H+L) (min X Ms Sr Prot)

21 Whole IgG Affinity-Purified Antibodies Ordering Technical Fluorescein FITC A=492, E=520 Cyanine Cy3 A=550, E=570 Rhodamine Red-X RRX A=570, E=590 Alexa Fluor 594 A=591, E=614 Alexa Fluor 647 A=651, E=667 Biotin-SP (long spacer) Horseradish Peroxidase Alkaline Phosphatase Telephone: +44 (0) Fax: +44 (0)

22 Whole IgG Affinity-Purified Antibodies Antibody Description Unconjugated DyLight 405 A=400, E=421 Coumarin AMCA A=350, E=450 Alexa Fluor 488 A=493, E=519 Whole IgG ANTI-HUMAN Mouse Anti-Human IgG (H+L) (min X Bov, Hrs, Ms Sr Prot) Mouse Anti-Human IgG, Fc γ fragment specific (min X Bov, Hrs, Ms Sr Prot) Mouse Anti-Human IgG, F(ab') 2 fragment specific (min X Bov, Hrs, Ms Sr Prot) Rabbit Anti-Human IgG (H+L) Rabbit Anti-Human IgG (H+L) (min X Ms Sr Prot) Rabbit Anti-Human IgG, Fc γ fragment specific Rabbit Anti-Human IgG, F(ab') 2 fragment specific Rabbit Anti-Human IgG + IgM (H+L) (min X Ms Sr Prot) Rabbit Anti-Human IgA + IgG + IgM (H+L) Rabbit Anti-Human IgM, Fc 5µ fragment specific (min X Ms Sr Prot) Rabbit Anti-Human Serum IgA, α chain specific Rabbit Anti-Human Lactoferrin Whole IgG ANTI-MOUSE Donkey Anti-Mouse IgG (H+L) (min X Bov, Ck, Gt, GP, Sy Hms, Hrs, Hu, Rb, Shp Sr Prot) Donkey Anti-Mouse IgG (H+L) (min X Bov, Ck, Gt, GP, Sy Hms, Hrs, Hu, Rb, Rat, Shp Sr Prot)** ** Caution: See page 6 (min X... Sr Prot) before selecting an antibody adsorbed against closely related species. * ML = Multiple Labelling (see Multiple Labelling on pages 6-7 for an explanation).

23 Whole IgG Affinity-Purified Antibodies Ordering Technical Fluorescein FITC A=492, E=520 Cyanine Cy3 A=550, E=570 Rhodamine Red-X RRX A=570, E=590 Alexa Fluor 594 A=591, E=614 Alexa Fluor 647 A=651, E=667 Biotin-SP (long spacer) Horseradish Peroxidase Alkaline Phosphatase Telephone: +44 (0) Fax: +44 (0)

24 Whole IgG Affinity-Purified Antibodies Antibody Description Unconjugated DyLight 405 A=400, E=421 Coumarin AMCA A=350, E=450 Alexa Fluor 488 A=493, E=519 Whole IgG ANTI-MOUSE Donkey Anti-Mouse IgM, µ chain specific Donkey Anti-Mouse IgM, µ chain specific (min X Hu, Bov, Hrs, Rat Sr Prot)** Goat Anti-Mouse IgG (H+L) Goat Anti-Mouse IgG (H+L) (min X Hu, Bov, Hrs Sr Prot) Goat Anti-Mouse IgG (H+L) (min X Hu, Bov, Hrs, Rb, Sw Sr Prot) Goat Anti-Mouse IgG (H+L) (min X Hu, Bov, Hrs, Rb, Rat Sr Prot)** Goat Anti-Mouse IgG, Light Chain Specific (min X Bov, Gt, Hrs, Hu, Rb, Rat, Shp Ig) Goat Anti-Mouse IgG, Fc γ fragment specific Goat Anti-Mouse IgG, Fc γ fragment specific (min X Hu, Bov, Hrs Sr Prot) Goat Anti-Mouse IgG, Fc γ Subclass 1 specific (min X Hu, Bov, Rb Sr Prot) Goat Anti-Mouse IgG, Fc γ Subclass 2a specific (min X Hu, Bov, Rb Sr Prot) Goat Anti-Mouse IgG, Fc γ Subclass 2b specific (min X Hu, Bov, Rb Sr Prot) Goat Anti-Mouse IgG, Fc γ Subclass 2c specific (min X Hu, Bov, Rb Sr Prot) Goat Anti-Mouse IgG, Fc γ Subclass 3 specific (min X Hu, Bov, Rb Sr Prot) Goat Anti-Mouse IgG (subclasses 1+2a+2b+3), Fc γ fragment specific (min X Hu, Bov, Rb Sr Prot) ** Caution: See page 6 (min X... Sr Prot) before selecting an antibody adsorbed against closely related species. * ML = Multiple Labelling (see Multiple Labelling on pages 6-7 for an explanation). This antibody reacts primarily with kappa light chains. It is not suitable for detection of primary antibodies with lambda light chains.

25 Whole IgG Affinity-Purified Antibodies Ordering Technical Fluorescein FITC A=492, E=520 Cyanine Cy3 A=550, E=570 Rhodamine Red-X RRX A=570, E=590 Alexa Fluor 594 A=591, E=614 Alexa Fluor 647 A=651, E=667 Biotin-SP (long spacer) Horseradish Peroxidase Alkaline Phosphatase Telephone: +44 (0) Fax: +44 (0)

26 Whole IgG Affinity-Purified Antibodies Antibody Description Unconjugated DyLight 405 A=400, E=421 Coumarin AMCA A=350, E=450 Alexa Fluor 488 A=493, E=519 Whole IgG ANTI-MOUSE Goat Anti-Mouse IgG, F(ab') 2 fragment specific Goat Anti-Mouse IgG, F(ab') 2 fragment specific (min X Hu, Bov, Hrs Sr Prot) Goat Anti-Mouse IgG + IgM (H+L) Goat Anti-Mouse IgG + IgM (H+L) (min X Hu, Bov, Hrs Sr Prot) Goat Anti-Mouse IgM, µ chain specific Goat Anti-Mouse IgM, µ chain specific (min X Hu, Bov, Hrs Sr Prot) Rabbit Anti-Mouse IgG (H+L) Rabbit Anti-Mouse IgG (H+L) (min X Hu Sr Prot) Rabbit Anti-Mouse IgG, Fc γ fragment specific Rabbit Anti-Mouse IgG, Fc γ fragment specific (min X Hu Sr Prot) Rabbit Anti-Mouse IgG, F(ab') 2 fragment specific Rabbit Anti-Mouse IgG, F(ab') 2 fragment specific (min X Hu Sr Prot) Rabbit Anti-Mouse IgG + IgM (H+L) Rabbit Anti-Mouse IgG + IgM (H+L) (min X Hu Sr Prot) Rabbit Anti-Mouse IgM, µ chain specific * ML = Multiple Labelling (see Multiple Labelling on pages 6-7 for an explanation).

27 Whole IgG Affinity-Purified Antibodies Ordering Technical Fluorescein FITC A=492, E=520 Cyanine Cy3 A=550, E=570 Rhodamine Red-X RRX A=570, E=590 Alexa Fluor 594 A=591, E=614 Alexa Fluor 647 A=651, E=667 Biotin-SP (long spacer) Horseradish Peroxidase Alkaline Phosphatase Telephone: +44 (0) Fax: +44 (0)

28 Whole IgG Affinity-Purified Antibodies Antibody Description Unconjugated DyLight 405 A=400, E=421 Coumarin AMCA A=350, E=450 Alexa Fluor 488 A=493, E=519 Whole IgG ANTI-MOUSE Rabbit Anti-Mouse IgM, µ chain specific (min X Hu Sr Prot) Rat Anti-Mouse IgG (H+L) (min X Hu, Bov, Hrs, Rb, Rat Sr Prot) Sheep Anti-Mouse IgG (H+L) Sheep Anti-Mouse IgG (H+L) (min X Hu, Bov, Hrs Sr Prot) Sheep Anti-Mouse IgG, Fc γ fragment specific (min X Hu, Bov, Hrs Sr Prot) Sheep Anti-Mouse IgG, F(ab') 2 fragment specific (min X Hu, Bov, Hrs Sr Prot) Whole IgG ANTI-RABBIT Donkey Anti-Rabbit IgG (H+L) (min X Bov, Ck, Gt, GP, Sy Hms, Hrs, Hu, Ms, Rat, Shp Sr Prot) Goat Anti-Rabbit IgG (H+L) Goat Anti-Rabbit IgG (H+L) (min X Hu Sr Prot) Goat Anti-Rabbit IgG (H+L) (min X Hu, Ms, Rat Sr Prot) Goat Anti-Rabbit IgG, Fc fragment specific Goat Anti-Rabbit IgG, Fc fragment specific (min X Hu Sr Prot) Goat Anti-Rabbit IgG, F(ab') 2 fragment specific Goat Anti-Rabbit IgG, F(ab') 2 fragment specific (min X Hu Sr Prot) * ML = Multiple Labelling (see Multiple Labelling on pages 6-7 for an explanation).

29 Whole IgG Affinity-Purified Antibodies Ordering Technical Fluorescein FITC A=492, E=520 Cyanine Cy3 A=550, E=570 Rhodamine Red-X RRX A=570, E=590 Alexa Fluor 594 A=591, E=614 Alexa Fluor 647 A=651, E=667 Biotin-SP (long spacer) Horseradish Peroxidase Alkaline Phosphatase Telephone: +44 (0) Fax: +44 (0)

30 Whole IgG Affinity-Purified Antibodies Antibody Description Unconjugated DyLight 405 A=400, E=421 Coumarin AMCA A=350, E=450 Alexa Fluor 488 A=493, E=519 Whole IgG ANTI-RABBIT Mouse Anti-Rabbit IgG (H+L) (min X Hu, Gt, Ms, Shp Sr Prot) IgG Fraction Monoclonal Mouse Anti-Rabbit IgG, Light Chain Specific (min X Bov, Gt, Ar Hms, Hrs, Hu, Ms, Rat, Shp Ig) Whole IgG ANTI-RAT Donkey Anti-Rat IgG (H+L) (min X Bov, Ck, Gt, GP, Sy Hms, Hrs, Hu, Rb, Shp Sr Prot) Donkey Anti-Rat IgG (H+L) (min X Bov, Ck, Gt, GP, Sy Hms, Hrs, Hu, Ms, Rb, Shp Sr Prot)** Goat Anti-Rat IgG (H+L) Goat Anti-Rat IgG (H+L) (min X Hu, Bov, Hrs Sr Prot) Goat Anti-Rat IgG (H+L) (min X Hu, Bov, Hrs, Rb Sr Prot) Goat Anti-Rat IgG (H+L) (min X Hu, Bov, Hrs, Ms, Rb Sr Prot)** Goat Anti-Rat IgG, Light Chain Specific (min X Bov, Gt, Hrs, Hu, Ms, Rb, Shp Ig) Goat Anti-Rat IgG, Fc γ fragment specific Goat Anti-Rat IgG, Fc γ fragment specific (min X Hu, Bov, Hrs Sr Prot) Goat Anti-Rat IgG, F(ab') 2 fragment specific Goat Anti-Rat IgG, F(ab') 2 fragment specific (min X Hu, Bov, Hrs Sr Prot) Goat Anti-Rat IgG + IgM (H+L) ** Caution: See page 6 (min X... Sr Prot) before selecting an antibody adsorbed against closely related species. * ML = Multiple Labelling (see Multiple Labelling on pages 6-7 for an explanation). This antibody reacts primarily with kappa light chains. It is not suitable for detection of primary antibodies with lambda light chains.

31 Whole IgG Affinity-Purified Antibodies Ordering Technical Fluorescein FITC A=492, E=520 Cyanine Cy3 A=550, E=570 Rhodamine Red-X RRX A=570, E=590 Alexa Fluor 594 A=591, E=614 Alexa Fluor 647 A=651, E=667 Biotin-SP (long spacer) Horseradish Peroxidase Alkaline Phosphatase Telephone: +44 (0) Fax: +44 (0)

32 Whole IgG Affinity-Purified Antibodies Antibody Description Unconjugated DyLight 405 A=400, E=421 Coumarin AMCA A=350, E=450 Alexa Fluor 488 A=493, E=519 Whole IgG ANTI-RAT Goat Anti-Rat IgG + IgM (H+L) (min X Hu, Bov, Hrs Sr Prot) Goat Anti-Rat IgM, µ chain specific Goat Anti-Rat IgM, µ chain specific (min X Hu, Bov, Hrs Sr Prot) Mouse Anti-Rat IgG (H+L) (min X Ms Sr Prot) Mouse Anti-Rat IgG (H+L) (min X Hu, Bov, Hrs, Ms, Gt, Rb Sr Prot) Mouse Anti-Rat IgG, Fc γ fragment specific (min X Hu, Bov, Hrs, Ms Sr Prot) Mouse Anti-Rat IgG, F(ab') 2 fragment specific (min X Hu, Bov, Hrs, Ms Sr Prot) Rabbit Anti-Rat IgG (H+L) Rabbit Anti-Rat IgG (H+L) (min X Hu Sr Prot) Rabbit Anti-Rat IgG, Fc γ fragment specific (min X Hu Sr Prot) Rabbit Anti-Rat IgG, F(ab') 2 fragment specific (min X Hu Sr Prot) Rabbit Anti-Rat IgG + IgM (H+L) Rabbit Anti-Rat IgG + IgM (H+L) (min X Hu Sr Prot) Rabbit Anti-Rat IgM, µ chain specific Rabbit Anti-Rat IgM, µ chain specific (min X Hu Sr Prot) * ML = Multiple Labelling (see Multiple Labelling on pages 6-7 for an explanation).

33 Whole IgG Affinity-Purified Antibodies Ordering Technical Fluorescein FITC A=492, E=520 Cyanine Cy3 A=550, E=570 Rhodamine Red-X RRX A=570, E=590 Alexa Fluor 594 A=591, E=614 Alexa Fluor 647 A=651, E=667 Biotin-SP (long spacer) Horseradish Peroxidase Alkaline Phosphatase Telephone: +44 (0) Fax: +44 (0)

34 Whole IgG Affinity-Purified Antibodies Antibody Description Unconjugated DyLight 405 A=400, E=421 Coumarin AMCA A=350, E=450 Alexa Fluor 488 A=493, E=519 Whole IgG ANTI-SHEEP Donkey Anti-Sheep IgG (H+L)*** Donkey Anti-Sheep IgG (H+L)*** (min X Ck, GP, Sy Hms, Hrs, Hu, Ms, Rb, Rat Sr Prot) IgG Fraction Monoclonal Mouse Anti-Sheep IgG, Light Chain Specific (min X Bov, Hrs, Hu, Ms, Rb, Rat Ig) Rabbit Anti-Sheep IgG (H+L)*** Rabbit Anti-Sheep IgG (H+L)*** (min X Hu Sr Prot) Rabbit Anti-Sheep IgG, Fc fragment specific*** (min X Hu Sr Prot) Rabbit Anti-Sheep IgG, F(ab') 2 fragment specific*** (min X Hu Sr Prot) Whole IgG ANTI-SWINE Goat Anti-Swine IgG (H+L) *** Warning: BSA and dry milk may contain IgG which will be recognised by this antibody. Use of BSA or dry milk to block or dilute this antibody may increase background and/or reduce secondary antibody titre. * ML = Multiple Labelling (see Multiple Labelling on pages 6-7 for an explanation).

35 Whole IgG Affinity-Purified Antibodies Ordering Technical Fluorescein FITC A=492, E=520 Cyanine Cy3 A=550, E=570 Rhodamine Red-X RRX A=570, E=590 Alexa Fluor 594 A=591, E=614 Alexa Fluor 647 A=651, E=667 Biotin-SP (long spacer) Horseradish Peroxidase Alkaline Phosphatase Telephone: +44 (0) Fax: +44 (0)

36 F(ab') 2 Fragment Affinity-Purified Antibodies Antibody Description Unconjugated Coumarin AMCA A=350, E=450 Alexa Fluor 488 A=493, E=519 F(ab') 2 Fragment ANTI-BOVINE Goat Anti-Bovine IgG (H+L)*** F(ab') 2 Fragment ANTI-CHICKEN Donkey Anti-Chicken IgY (IgG) (H+L) (min X Bov, Gt, GP, Sy Hms, Hrs, Hu, Ms, Rb, Rat, Shp Sr Prot) Rabbit Anti-Chicken IgY (IgG) (H+L) F(ab') 2 Fragment ANTI-GOAT Donkey Anti-Goat IgG (H+L)*** (min X Ck, GP, Sy Hms, Hrs, Hu, Ms, Rb, Rat Sr Prot) Rabbit Anti-Goat IgG (H+L)*** Rabbit Anti-Goat IgG (H+L)*** (min X Hu Sr Prot) Rabbit Anti-Goat IgG, Fc fragment specific*** Rabbit Anti-Goat IgG, Fc fragment specific*** (min X Hu Sr Prot) Rabbit Anti-Goat IgG, F(ab') 2 fragment specific*** Rabbit Anti-Goat IgG, F(ab') 2 fragment specific*** (min X Hu Sr Prot) F(ab') 2 Fragment ANTI-GUINEA PIG Donkey Anti-Guinea Pig IgG (H+L) (min X Bov, Ck, Gt, Sy Hms, Hrs, Hu, Ms, Rb, Rat, Shp Sr Prot) Goat Anti-Guinea Pig IgG (H+L) *** Warning: BSA and dry milk may contain IgG which will be recognised by this antibody. Use of BSA or dry milk to block or dilute this antibody may increase background and/or reduce secondary antibody titre. * ML = Multiple Labelling (see Multiple Labelling on pages 6-7 for an explanation). IgY is the original designation for the IgG-like protein found in both serum and egg yolk.

37 F(ab') 2 Fragment Affinity-Purified Antibodies Ordering cuserv@jireurope.com Technical tech@jireurope.com Fluorescein FITC A=492, E=520 Cyanine Cy3 A=550, E=570 Rhodamine Red-X RRX A=570, E=590 Alexa Fluor 594 A=591, E=614 Alexa Fluor 647 A=651, E=667 Biotin-SP (long spacer) Horseradish Peroxidase Alkaline Phosphatase Telephone: +44 (0) Fax: +44 (0)

38 F(ab') 2 Fragment Affinity-Purified Antibodies Antibody Description Unconjugated Coumarin AMCA A=350, E=450 Alexa Fluor 488 A=493, E=519 F(ab') 2 Fragment ANTI-HAMSTER (Syrian Hamster) Goat Anti-Syrian Hamster IgG (H+L) (min X Bov, Hrs, Hu, Ms, Rb, Rat Sr Prot) Rabbit Anti-Syrian Hamster IgG (H+L) F(ab') 2 Fragment ANTI-HORSE Goat Anti-Horse IgG (H+L) F(ab') 2 Fragment ANTI-HUMAN Donkey Anti-Human IgG (H+L) (min X Bov, Ck, Gt, GP, Sy Hms, Hrs, Ms, Rb, Rat, Shp Sr Prot) Donkey Anti-Human IgG, Fc γ fragment specific (min X Bov, Hrs, Ms Sr Prot) Donkey Anti-Human IgM, Fc 5µ fragment specific (min X Bov, Hrs Sr Prot) Goat Anti-Human IgG (H+L) Goat Anti-Human IgG (H+L) (min X Bov, Hrs, Ms Sr Prot) Goat Anti-Human IgG, Fc γ fragment specific Goat Anti-Human IgG, Fc γ fragment specific (min X Bov, Hrs, Ms Sr Prot) Goat Anti-Human IgG, F(ab') 2 fragment specific Goat Anti-Human IgG, F(ab') 2 fragment specific (min X Bov, Hrs, Ms Sr Prot) Goat Anti-Human IgG + IgM (H+L) (min X Bov Sr Prot) Goat Anti-Human IgA + IgG + IgM (H+L) * ML = Multiple Labelling (see Multiple Labelling on pages 6-7 for an explanation).

39 F(ab') 2 Fragment Affinity-Purified Antibodies Ordering cuserv@jireurope.com Technical tech@jireurope.com Fluorescein FITC A=492, E=520 Cyanine Cy3 A=550, E=570 Rhodamine Red-X RRX A=570, E=590 Alexa Fluor 594 A=591, E=614 Alexa Fluor 647 A=651, E=667 Biotin-SP (long spacer) Horseradish Peroxidase Alkaline Phosphatase Telephone: +44 (0) Fax: +44 (0)

40 F(ab') 2 Fragment Affinity-Purified Antibodies Antibody Description Unconjugated Coumarin AMCA A=350, E=450 Alexa Fluor 488 A=493, E=519 F(ab') 2 Fragment ANTI-HUMAN Goat Anti-Human IgM, Fc 5µ fragment specific (min X Bov Sr Prot) Goat Anti-Human Serum IgA, α chain specific Rabbit Anti-Human IgG (H+L) Rabbit Anti-Human IgG, Fc γ fragment specific Rabbit Anti-Human IgM, Fc 5µ fragment specific F(ab') 2 Fragment ANTI-MOUSE Donkey Anti-Mouse IgG (H+L) (min X Bov, Ck, Gt, GP, Sy Hms, Hrs, Hu, Rb, Shp Sr Prot) Donkey Anti-Mouse IgG (H+L) (min X Bov, Ck, Gt, GP, Sy Hms, Hrs, Hu, Rb, Rat, Shp Sr Prot)** Donkey Anti-Mouse IgM, µ chain specific Goat Anti-Mouse IgG (H+L) Goat Anti-Mouse IgG (H+L) (min X Hu, Bov, Hrs Sr Prot) Goat Anti-Mouse IgG (H+L) (min X Hu, Bov, Hrs, Rb, Sw Sr Prot) Goat Anti-Mouse IgG, Fc γ fragment specific Goat Anti-Mouse IgG, Fc γ fragment specific (min X Hu, Bov, Hrs Sr Prot) Goat Anti-Mouse IgG, F(ab') 2 fragment specific ** Caution: See page 6 (min X... Sr Prot) before selecting an antibody adsorbed against closely related species. * ML = Multiple Labelling (see Multiple Labelling on pages 6-7 for an explanation).

41 F(ab') 2 Fragment Affinity-Purified Antibodies Ordering cuserv@jireurope.com Technical tech@jireurope.com Fluorescein FITC A=492, E=520 Cyanine Cy3 A=550, E=570 Rhodamine Red-X RRX A=570, E=590 Alexa Fluor 594 A=591, E=614 Alexa Fluor 647 A=651, E=667 Biotin-SP (long spacer) Horseradish Peroxidase Alkaline Phosphatase Telephone: +44 (0) Fax: +44 (0)

42 F(ab') 2 Fragment Affinity-Purified Antibodies Antibody Description Unconjugated Coumarin AMCA A=350, E=450 Alexa Fluor 488 A=493, E=519 F(ab') 2 Fragment ANTI-MOUSE Goat Anti-Mouse IgG, F(ab') 2 fragment specific (min X Hu, Bov, Hrs Sr Prot) Goat Anti-Mouse IgG + IgM (H+L) (min X Hu, Bov, Hrs Sr Prot) Goat Anti-Mouse IgM, µ chain specific Goat Anti-Mouse IgM, µ chain specific (min X Hu, Bov, Hrs Sr Prot) Rabbit Anti-Mouse IgG (H+L) Rabbit Anti-Mouse IgG (H+L) (min X Hu Sr Prot) Rabbit Anti-Mouse IgG, Fc γ fragment specific (min X Hu Sr Prot) Rabbit Anti-Mouse IgG, F(ab') 2 fragment specific (min X Hu Sr Prot) F(ab') 2 Fragment ANTI-RABBIT Donkey Anti-Rabbit IgG (H+L) (min X Bov, Ck, Gt, GP, Sy Hms, Hrs, Hu, Ms, Rat, Shp Sr Prot) Goat Anti-Rabbit IgG (H+L) Goat Anti-Rabbit IgG (H+L) (min X Hu Sr Prot) Goat Anti-Rabbit IgG (H+L) (min X Hu, Ms, Rat Sr Prot) Goat Anti-Rabbit IgG, Fc fragment specific (min X Hu Sr Prot) Goat Anti-Rabbit IgG, F(ab') 2 fragment specific (min X Hu Sr Prot) * ML = Multiple Labelling (see Multiple Labelling on pages 6-7 for an explanation).

43 F(ab') 2 Fragment Affinity-Purified Antibodies Ordering cuserv@jireurope.com Technical tech@jireurope.com Fluorescein FITC A=492, E=520 Cyanine Cy3 A=550, E=570 Rhodamine Red-X RRX A=570, E=590 Alexa Fluor 594 A=591, E=614 Alexa Fluor 647 A=651, E=667 Biotin-SP (long spacer) Horseradish Peroxidase Alkaline Phosphatase Telephone: +44 (0) Fax: +44 (0)

44 F(ab') 2 Fragment Affinity-Purified Antibodies Antibody Description Unconjugated Coumarin AMCA A=350, E=450 Alexa Fluor 488 A=493, E=519 F(ab') 2 Fragment ANTI-RAT Donkey Anti-Rat IgG (H+L) (min X Bov, Ck, Gt, GP, Sy Hms, Hrs, Hu, Rb, Shp Sr Prot) Donkey Anti-Rat IgG (H+L) (min X Bov, Ck, Gt, GP, Sy Hms, Hrs, Hu, Ms, Rb, Shp Sr Prot)** Goat Anti-Rat IgG (H+L) Goat Anti-Rat IgG (H+L) (min X Hu, Bov, Hrs Sr Prot) Goat Anti-Rat IgG (H+L) (min X Hu, Bov, Hrs, Rb Sr Prot) Goat Anti-Rat IgG, Fc γ fragment specific (min X Hu, Bov, Hrs Sr Prot) Goat Anti-Rat IgG, F(ab') 2 fragment specific (min X Hu, Bov, Hrs Sr Prot) Goat Anti-Rat IgG + IgM (H+L) (min X Hu, Bov, Hrs Sr Prot) Goat Anti-Rat IgM, µ chain specific (min X Hu, Bov, Hrs Sr Prot) Mouse Anti-Rat IgG (H+L) (min X Hu, Bov, Hrs, Ms, Gt, Rb Sr Prot) Rabbit Anti-Rat IgG (H+L) (min X Hu Sr Prot) F(ab') 2 Fragment ANTI-SHEEP Donkey Anti-Sheep IgG (H+L)*** (min X Ck, GP, Sy Hms, Hrs, Hu, Ms, Rb, Rat Sr Prot) Rabbit Anti-Sheep IgG (H+L)*** *** Warning: BSA and dry milk may contain IgG which will be recognised by this antibody. Use of BSA or dry milk to block or dilute this antibody may increase background and/or reduce secondary antibody titre. ** Caution: See page 6 (min X... Sr Prot) before selecting an antibody adsorbed against closely related species. * ML = Multiple Labelling (see Multiple Labelling on pages 6-7 for an explanation).

45 F(ab') 2 Fragment Affinity-Purified Antibodies Ordering cuserv@jireurope.com Technical tech@jireurope.com Fluorescein FITC A=492, E=520 Cyanine Cy3 A=550, E=570 Rhodamine Red-X RRX A=570, E=590 Alexa Fluor 594 A=591, E=614 Alexa Fluor 647 A=651, E=667 Biotin-SP (long spacer) Horseradish Peroxidase Alkaline Phosphatase Telephone: +44 (0) Fax: +44 (0)

46 Monovalent Fab Fragment Affinity-Purified Antibodies for Blocking and for Double Labelling Primary Antibodies from the Same Host Species Monovalent Fab fragments of affinity-purified secondary antibodies are offered to cover (block) the surface of immunoglobulins for double labelling primary antibodies from the same host species, or to block endogenous immunoglobulins in tissue sections or on cell surfaces. They can be used for these purposes because Fab fragments have a single antigen binding site (i.e. they are monovalent). In contrast, divalent antibodies (whole IgG and F(ab') 2 fragments) have two antigen binding sites. After labelling the first primary antibody, some antigen binding sites on the first secondary antibody may remain open which could capture the second primary antibody introduced in a subsequent step. Consequently, it will appear as overlapping labelling, even though there may not be overlapping antigens. Therefore, divalent antibodies should not be used for blocking or for double labelling two primary antibodies from the same species. For selected literature references see Wessel and McClay, J. Histochem. Cytochem , 703; Franzusoff et al., J. Cell Biology , 27; Lewis Carl et al., J. Histochem. Cytochem , 1273; and Negoescu et al., J. Histochem. Cytochem , 433. Monovalent Fab secondary antibodies are not necessary when primary antibodies from the same host species are different classes of immunoglobulins, such as IgG and IgM; or different subclasses of IgG, such as Mouse IgG1 and Mouse IgG2a. In these cases it is much easier and more effective to use class-specific, or subclass-specific antibodies respectively, to distinguish between the two primary antibodies from the same species. To block endogenous immunoglobulins on cells or tissue sections, incubate with an excess (20-40 μg/ml) of unconjugated Fab antibody just after blocking with 5% normal serum. Samples with high levels of endogenous immunoglobulins may require up to 100 µg/ml of unconjugated Fab. To avoid a possible displacement of the Fab antibody by the labelled secondary antibody, a light post-fixation with glutaraldehyde may be necessary, provided that it does not affect antigenicity of the target proteins. Fab antibodies are not effective for blocking immunoglobulins in Western blotting or ELISA applications. Antibody Description Donkey Anti-Goat IgG (H+L)*** Rabbit Anti-Goat IgG (H+L)*** Goat Anti-Human IgG (H+L) Goat Anti-Human IgM, Fc 5μ fragment specific Donkey Anti-Mouse IgG (H+L) Goat Anti-Mouse IgG (H+L) Goat Anti-Mouse IgM, μ chain specific Rabbit Anti-Mouse IgG (H+L) Donkey Anti-Rabbit IgG (H+L) Goat Anti-Rabbit IgG (H+L) Donkey Anti-Rat IgG (H+L) Goat Anti-Rat IgG (H+L) Rabbit Anti-Sheep IgG (H+L)*** 46 *** Warning: BSA and dry milk may contain IgG which will be recognised by this antibody. Use of BSA or dry milk to block or dilute this antibody may increase background and/or reduce secondary antibody titre.

47 Ordering Technical Monovalent Fab Fragment Affinity-Purified Antibodies for Blocking and for Double Labelling Primary Antibodies from the Same Host Species Unconjugated Alexa Fluor 488 A=493, E=519 Fluorescein FITC A=492, E=520 Cyanine Cy3 A=550, E=570 Rhodamine Red-X RRX A=570, E=590 Alexa Fluor 594 A=591, E=614 Alexa Fluor 647 A=651, E=667 Biotin-SP (long spacer) Telephone: +44 (0) Fax: +44 (0)

48 Monovalent Fab Fragment Affinity-Purified Antibodies for Blocking and for Double Labelling Primary Antibodies from the Same Host Species Fab Fragments for Blocking and Double Labelling of Primary Antibodies from the Same Host Species The following examples show some of the possible protocols used for double labelling two unconjugated primary antibodies from the same host species. The success of these experimental designs will require some empirical manipulations. In each case, it is important to label the less abundant primary antibody first. Trying different concentrations of reagents in each step or switching the labelling sequence of the two antigens may influence the outcome. Blocking with an appropriate normal serum between certain steps may also help to reduce background. To avoid a possible displacement of the Fab antibody by the labelled secondary antibody, a light post-fixation with glutaraldehyde may be necessary, provided that it does not affect antigenicity of the target proteins. Example A: Use of conjugated Fab fragments for labelling and blocking. KEY Rabbit anti-antigen X Goat anti-rabbit IgG (H+L) Rabbit anti-antigen Y Alexa Fluor 488 Fab fragment Goat anti-rabbit IgG (H+L) Rhodamine Red-X Important note: Monovalent Fab fragments have not been adsorbed to remove cross-reactivities to other species. If the cells/tissues under study have endogenous immunoglobulins, using Example A or B may create background. In this case Example C should be used (see page 50). X Y X Y X Y 1. Incubate with the first primary antibody, in this example rabbit anti-antigen X. Wash. 2. Incubate with excess Probe I-conjugated secondary antibody, in this example Alexa Fluor 488-Fab fragment goat anti-rabbit IgG (H+L). Wash. 3. Incubate with the second primary antibody, rabbit anti-antigen Y, followed by Probe II-conjugated secondary antibody, in this example Rhodamine Red-X-goat anti-rabbit IgG (H+L). Wash. Caution: This protocol may be subject to the following problems: (1) Example A may require a high concentration of conjugated Fab to achieve effective blocking of the first primary antibody. If this results in unacceptable background, try a lower concentration of the conjugated Fab, followed by further blocking with unconjugated Fab. 48 (2) If small aggregates of conjugated Fab are bound to the first primary antibody, they may act as divalent or polyvalent antibodies and capture some of the second primary antibodies, which would result in overlapping detection of antigens.

49 Ordering Technical Monovalent Fab Fragment Affinity-Purified Antibodies for Blocking and for Double Labelling Primary Antibodies from the Same Host Species Example B: Use of unconjugated Fab fragments to cover the first primary antibody, presenting it KEY as a different species. Rabbit anti-antigen X Mouse anti-rabbit IgG (H+L) (min X Hu, Gt, Ms, Shp Sr Prot) Rabbit anti-antigen Y Fab fragment Goat anti-rabbit IgG (H+L) Alexa Fluor 488 Rhodamine Red-X Mouse anti-goat IgG (H+L)(min X Hu, Ms, Rb Sr Prot) Y Y Y Y Y X X X X X 1. Incubate with the first primary antibody, in this example rabbit anti-antigen X. Wash. 2. Incubate with an excess of unconjugated Fab antibody against the host species of the primary antibody, in this example unconjugated Fab fragment goat anti-rabbit IgG (H+L). This presents the rabbit IgG as goat Fab. Wash. 4. Incubate with the second primary antibody, in this example rabbit anti-antigen Y. Wash. 5. Incubate with Probe II-conjugated to the second secondary antibody, that does not recognise the host species of either the Fab antibody used in step 2 or the tertiary antibody used in step 3. In this example, Rhodamine Red-X-mouse anti-rabbit IgG (H+L) (min X Hu, Gt, Ms and Shp Sr Prot) was used. Wash. 3. Incubate with Probe I-conjugated tertiary antibody directed against the host species of the Fab antibody. The tertiary antibody must not recognise the host species of either the primary antibodies or the second secondary antibody. This example used Alexa Fluor 488-mouse anti-goat IgG (H+L) (min X Hu, Ms, Rb Sr Prot). Wash. Telephone: +44 (0) Fax: +44 (0)

50 Monovalent Fab Fragment Affinity-Purified Antibodies for Blocking and for Double Labelling Primary Antibodies from the Same Host Species Example C: KEY Use of unconjugated Fab fragments for blocking after the first secondary antibody step. Rabbit anti-antigen X Alexa Fluor 488 Rabbit anti-antigen Y Fab fragment Goat anti-rabbit IgG (H+L) Rhodamine Red-X Rabbit IgG from normal rabbit serum Goat anti-rabbit IgG (H+L) (min X Hu, Ms, Rat, Sr Prot) Y Y Y Y Y Y X X X X X X 1. Incubate with the first primary antibody, in this example rabbit anti-antigen X. Wash. 2. Incubate with Probe I-conjugated secondary antibody, in this example Alexa Fluor 488-goat anti-rabbit IgG (H+L) (min X Hu, Ms, Rat Sr Prot). Wash. 3. Incubate with normal serum from the same host species as the primary antibodies, in this example normal rabbit serum. The purpose of this step is to saturate open binding sites on the first secondary antibody with IgG so that they cannot capture the second primary antibody. Wash. 4. Incubate with an excess of unconjugated Fab antibody against the host species of the primary antibodies, in this example Fab goat anti-rabbit IgG (H+L). The host species of the Fab antibody should be the same as the host species of the conjugated secondary antibody. This step covers the rabbit IgG so that the second secondary antibody will not bind to it. Wash. 5. Incubate with the second primary antibody, in this example rabbit anti-antigen Y. Wash. 6. Incubate with Probe II conjugated to the same secondary antibody as used in step 2, in this example Rhodamine Red-X-goat anti-rabbit IgG (H+L)(min X Hu, Ms, Rat Sr Prot). Wash. 50

51 Ordering Technical Monovalent Fab Fragment Affinity-Purified Antibodies for Blocking and for Double Labelling Primary Antibodies from the Same Host Species Examples D and E illustrate multiple labelling of primary antibodies from the same host species when one or more of the primaries is directly labelled and does not require a secondary antibody. If it is necessary to incubate with the labelled primary antibody first (Example D), blocking with a monovalent Fab antibody is required to prevent the secondary antibody used in a subsequent step from binding to the labelled primary antibody. Incubating with the unlabelled primary antibody first (Example E) will obviate the need for blocking with a monovalent Fab antibody. KEY Rabbit anti-antigen X Rabbit anti-antigen Y Fab fragment Goat anti-rabbit IgG (H+L) Goat anti-rabbit IgG (H+L) Alexa Fluor 488 Rhodamine Red-X Example D: Use of unconjugated Fab fragments for detection of one unlabelled and one or more labelled primary antibodies Y Y Y Y X X X X 1. Incubate with Probe I-conjugated to the first primary antibody, in this example Alexa Fluor 488-rabbit antiantigen X. Wash. 2. Incubate with an excess of unconjugated Fab goat anti-rabbit IgG (H+L). Wash. 3. Incubate with the unconjugated primary antibody, in this example rabbit anti-antigen Y. Wash. 4. Incubate with Probe II-conjugated secondary antibody, in this example Rhodamine Red-X-goat anti-rabbit IgG (H+L). Wash. Telephone: +44 (0) Fax: +44 (0)

52 Monovalent Fab Fragment Affinity-Purified Antibodies for Blocking and for Double Labelling Primary Antibodies from the Same Host Species Example E: Detection of one unlabelled and one or more labelled primary antibodies without the use of Fab fragments. KEY Rabbit anti-antigen X Alexa Fluor 488 Rabbit anti-antigen Y Rhodamine Red-X Goat anti-rabbit IgG (H+L) Rabbit IgG from normal rabbit serum Y Y Y Y X X X X 1. Incubate with the first primary antibody, in this example rabbit anti-antigen X. Wash. 2. Incubate with Probe I-conjugated secondary antibody, in this example Alexa Fluor 488-goat anti-rabbit IgG (H+L). Wash. 3. Incubate with normal serum from the host species of the primary antibody, in this example normal rabbit serum. Wash. 4. Incubate with Probe II-conjugated primary antibody, in this example Rhodamine Red-X-rabbit anti-antigen Y. Wash. 52

53 Secondary Antibodies for Flow Cytometry Ordering Technical Secondary Antibodies for Flow Cytometry For flow cytometry we offer three fluorescent proteins (R-PE, APC and PerCP) conjugated to many highly adsorbed secondary antibodies, streptavidin and purified immunoglobulin controls. The table of Secondary Antibodies for Flow Cytometry lists all of the antibodies, purified immunoglobulin controls and streptavidin to which we conjugate these proteins. Also shown in this table are the same highly adsorbed antibodies and purified immunoglobulins conjugated to Biotin-SP and fluorescent dyes appropriate for flow cytometry (Alexa Fluor 488, FITC and Alexa Fluor 647). Note that many antibodies listed elsewhere in tables of Whole IgG and F(ab') 2 Fragments also can be used for flow cytometry. Phycoerythrin (R-PE) and allophycocyanin (APC) are among several kinds of lightharvesting phycobiliproteins found in red, blue-green and cryptomonad algae. We offer R-PE, the form found in red macrophytic algae (seaweed). APC is isolated from the bluegreen alga Spirulina and is chemically cross-linked for stability. After phycobiliproteins are conjugated to secondary antibodies, there is little fluorescence quenching, which results in conjugates of high specific fluorescence compared with conventional fluorophoreantibody conjugates. R-PE, PerCP and APC can be excited by light over a wide range of the visible spectrum (Figure 6), are highly water soluble, have relatively low isoelectric points and lack potentially sticky carbohydrates. PerCP is a fluorescent peridinin-chlorophyll-protein complex isolated from dinoflagellates. We offer the form found in Dinophyceae sp. with a molecular weight of about 35.5 kda. It has a broad spectrum of excitation with a main peak at 482 nm and a long Stokes shift to an emission peak at 677 nm (Figure 6). PerCP, Alexa Fluor 488 (or FITC) and R-PE are excited at 488 nm with an argon laser (Figure 6) and thus can be used for one-, two- and three-colour analyses with single-laser flow cytometers. APC and Alexa Fluor 647 are excited at 633 nm (Figure 6) to give a fourth colour with dual-laser flow cytometers. It should be noted that the relatively high molecular weights of PerCP, R-PE and APC may preclude their use in procedures requiring good penetration into cells and tissues. They are predominantly intended for surface labelling of cells for flow cytometry. Figure 6. Excitation and emission spectra of Alexa Fluor 488/FITC (green), R-PE (red), PerCP (blue), Alexa Fluor 647 (black) and APC (brown). Peak heights were normalised after the spectra were obtained with an M-series spectrofluorometer system from Photon Technology International, Inc. Excitation Fluorescence Wavelength (nm) Wavelength (nm) Figure 6. Telephone: +44 (0) Fax: +44 (0)

54 Secondary Antibodies for Flow Cytometry Antibody Description Unconjugated Alexa Fluor 488 A=493, E=519 F(ab') 2 Fragment Donkey Anti-Chicken IgY (IgG)(H+L) (min X Bov, Gt, GP, Sy Hms, Hrs, Hu, Ms, Rb, Rat, Shp Sr Prot) F(ab') 2 Fragment Donkey Anti-Goat IgG (H+L)*** (min X Ck, GP, Sy Hms, Hrs, Hu, Ms, Rb, Rat Sr Prot) F(ab') 2 Fragment Donkey Anti-Guinea Pig IgG (H+L) (min X Bov, Ck, Gt, Sy Hms, Hrs, Hu, Ms, Rb, Rat, Shp Sr Prot) Whole IgG Goat Anti-Armenian Hamster IgG (H+L) (min X Bov, Hu, Ms, Rb, Rat Sr Prot)** F(ab') 2 Fragment Donkey Anti-Human IgG (H+L) (min X Bov, Ck, Gt, GP, Sy Hms, Hrs, Ms, Rb, Rat, Shp Sr Prot) F(ab') 2 Fragment Donkey Anti-Human IgG, Fc γ fragment specific (min X Bov, Hrs, Ms Sr Prot) F(ab') 2 Fragment Donkey Anti-Human IgM, Fc 5μ fragment specific (min X Bov, Hrs Sr Prot) F(ab') 2 Fragment Goat Anti-Human IgG (H+L) (min X Bov, Hrs, Ms Sr Prot) F(ab') 2 Fragment Goat Anti-Human IgG, Fc γ fragment specific (min X Bov, Hrs, Ms Sr Prot) Whole IgG Goat Anti-Human IgG, Fc γ fragment specific (min X Bov, Hrs, Ms Sr Prot) F(ab') 2 Fragment Goat Anti-Human IgG, Fc γ fragment specific (min X Bov, Ms, Rb Sr Prot) F(ab') 2 Fragment Goat Anti-Human IgG, F(ab') 2 fragment specific (min X Bov, Hrs, Ms Sr Prot) F(ab') 2 Fragment Goat Anti-Human IgG + IgM (H+L) (min X Bov Sr Prot) Whole IgG Goat Anti-Human IgA, α chain specific F(ab') 2 Fragment Donkey Anti-Mouse IgG (H+L) (min X Bov, Ck, Gt, GP, Sy Hms, Hrs, Hu, Rb, Shp Sr Prot) *** Warning: BSA and dry milk may contain IgG which will be recognised by this antibody. Use of BSA or dry milk to block or dilute this antibody may increase background and/or reduce secondary antibody titre. ** Caution: See page 6 (min X... Sr Prot) before selecting an antibody adsorbed against closely related species. * ML = Multiple Labelling (see Multiple Labelling on pages 6-7 for an explanation). IgY is the original designation for the IgG-like protein found in both serum and egg yolk.

55 Secondary Antibodies for Flow Cytometry Ordering Technical Fluorescein FITC A=488, E=520 Phycoerythrin R-PE A=488, E=580 Allophycocyanin APC A=650, E=660 Alexa Fluor 647 A=651, E=667 PerCP A=488, E=675 Biotin-SP (long spacer) Telephone: +44 (0) Fax: +44 (0)

56 Secondary Antibodies for Flow Cytometry Antibody Description Unconjugated Alexa Fluor 488 A=493, E=519 F(ab') 2 Fragment Donkey Anti-Mouse IgG (H+L) (min X Bov, Ck, Gt, GP, Sy Hms, Hrs, Hu, Rb, Rat, Shp Sr Prot)** F(ab') 2 Fragment Goat Anti-Mouse IgG (H+L) (min X Hu, Bov, Hrs, Rb, Sw Sr Prot) F(ab') 2 Fragment Goat Anti-Mouse IgG Fc γ fragment specific (min X Hu, Bov, Hrs Sr Prot) Whole IgG Goat Anti-Mouse IgG (subclasses 1+2a+2b+3), Fc γ fragment specific (min X Hu, Bov, Rb Sr Prot) Whole IgG Goat Anti-Mouse IgG, Fc γ subclass 1 specific (min X Hu, Bov, Rb Sr Prot) Whole IgG Goat Anti-Mouse IgG, Fc γ subclass 2a specific (min X Hu, Bov, Rb Sr Prot) Whole IgG Goat Anti-Mouse IgG, Fc γ subclass 2b specific (min X Hu, Bov, Rb Sr Prot) Whole IgG Goat Anti-Mouse IgG, Fc γ subclass 2c specific (min X Hu, Bov, Rb Sr Prot) Whole IgG Goat Anti-Mouse IgG, Fc γ subclass 3 specific (min X Hu, Bov, Rb Sr Prot) F(ab') 2 Fragment Goat Anti-Mouse IgG, F(ab') 2 fragment specific (min X Hu, Bov, Hrs Sr Prot) F(ab') 2 Fragment Goat Anti-Mouse IgG + IgM (H+L) (min X Hu, Bov, Hrs Sr Prot) F(ab') 2 Fragment Goat Anti-Mouse IgM, μ chain specific (min X Hu, Bov, Hrs Sr Prot) F(ab') 2 Fragment Donkey Anti-Rabbit IgG (H+L) (min X Bov, Ck, Gt, GP, Sy Hms, Hrs, Hu, Ms, Rat, Shp Sr Prot) F(ab') 2 Fragment Goat Anti-Rabbit IgG (H+L) (min X Hu, Ms, Rat Sr Prot) F(ab') 2 Fragment Donkey Anti-Rat IgG (H+L) (min X Bov, Ck, Gt, GP, Sy Hms, Hrs, Hu, Rb, Shp Sr Prot) ** Caution: See page 6 (min X... Sr Prot) before selecting an antibody adsorbed against closely related species. * ML = Multiple Labelling (see Multiple Labelling on pages 6-7 for an explanation).

57 Secondary Antibodies for Flow Cytometry Ordering Technical Fluorescein FITC A=488, E=520 Phycoerythrin R-PE A=488, E=580 Allophycocyanin APC A=650, E=660 Alexa Fluor 647 A=651, E=667 PerCP A=488, E=675 Biotin-SP (long spacer) Telephone: +44 (0) Fax: +44 (0)

58 Secondary Antibodies for Flow Cytometry Antibody Description Unconjugated Alexa Fluor 488 A=493, E=519 F(ab') 2 Fragment Donkey Anti-Rat IgG (H+L) (min X Bov, Ck, Gt, GP, Sy Hms, Hrs, Hu, Ms, Rb, Shp Sr Prot)** F(ab') 2 Fragment Goat Anti-Rat IgG (H+L) (min X Hu, Bov, Hrs, Rb Sr Prot) F(ab') 2 Fragment Goat Anti-Rat IgG, Fc γ fragment specific (min X Hu, Bov, Hrs Sr Prot) F(ab') 2 Fragment Goat Anti-Rat IgG, F(ab') 2 fragment specific (min X Hu, Bov, Hrs Sr Prot) F(ab') 2 Fragment Goat Anti-Rat IgM, μ chain specific (min X Hu, Bov, Hrs Sr Prot) F(ab') 2 Fragment Donkey Anti-Sheep IgG (H+L)*** (min X Ck, GP, Sy Hms, Hrs, Hu, Ms, Rb, Rat Sr Prot) IgG Controls and Streptavidin Conjugates for Flow Cytometry Streptavidin and IgG Controls Unconjugated Alexa Fluor 488 A=493, E=519 Streptavidin ChromPure Goat IgG, whole molecule ChromPure Goat IgG, F(ab') 2 fragment ChromPure Donkey IgG, F(ab') 2 fragment mg *** Warning: BSA and dry milk may contain IgG which will be recognised by this antibody. Use of BSA or dry milk to block or dilute this antibody may increase background and/or reduce secondary antibody titre. ** Caution: See page 6 (min X... Sr Prot) before selecting an antibody adsorbed against closely related species. * ML = Multiple Labelling (see Multiple Labelling on pages 6-7 for an explanation).

59 Secondary Antibodies for Flow Cytometry Ordering Technical Fluorescein FITC A=488, E=520 Phycoerythrin R-PE A=488, E=580 Allophycocyanin APC A=650, E=660 Alexa Fluor 647 A=651, E=667 PerCP A=488, E=675 Biotin-SP (long spacer) Fluorescein FITC/DTAF A=488, E=520 Phycoerythrin R-PE A=488, E=580 Allophycocyanin APC A=650, E=660 Alexa Fluor 647 A=651, E=667 PerCP A=488, E=675 Biotin-SP (long spacer) (DTAF) (FITC) (FITC) (FITC) Telephone: +44 (0) Fax: +44 (0)

60 Anti-Mouse IgG Subclass Specific Antibodies These antibodies have been extensively adsorbed against other mouse IgG subclasses. They are intended for distinguishing between two or more different subclasses of mouse IgG in multiple-labelling experiments, or for mouse IgG subclass determination. They are not necessary for general detection of mouse monoclonal antibodies in single-labelling experiments or in multiple-labelling experiments involving one mouse monoclonal and primary antibodies from other species. They have been adsorbed against human, bovine and rabbit serum proteins to minimise interfering cross-reactivities with tissue immunoglobulins, adherent bovine IgG on cultured cells and rabbit primary antibodies. Antibody Description Unconjugated DyLight 405 A=400, E=421 Alexa Fluor 488 A=493, E=519 Whole IgG Goat Anti-Mouse IgG, Fc γ Subclass 1 specific (min X Hu, Bov, Rb Sr Prot) Whole IgG Goat Anti-Mouse IgG, Fc γ Subclass 2a specific (min X Hu, Bov, Rb Sr Prot) Whole IgG Goat Anti-Mouse IgG, Fc γ Subclass 2b specific (min X Hu, Bov, Rb Sr Prot) Whole IgG Goat Anti-Mouse IgG, Fc γ Subclass 2c specific (min X Hu, Bov, Rb Sr Prot) Whole IgG Goat Anti-Mouse IgG, Fc γ Subclass 3 specific (min X Hu, Bov, Rb Sr Prot) * ML = Multiple Labelling (see Multiple Labelling on pages 6-7 for an explanation).

61 Anti-Mouse IgG Subclass Specific Antibodies Ordering Technical Fluorescein FITC A=492, E=520 Cyanine Cy3 A=550, E=570 Rhodamine Red-X RRX A=570, E=590 Alexa Fluor 594 A=591, E=614 Alexa Fluor 647 A=651, E=667 Biotin-SP (long spacer) Horseradish Peroxidase Alkaline Phosphatase ml Telephone: +44 (0) Fax: +44 (0)

62 Anti-IgG, Light Chain Specific Antibodies for Western Blotting after IP Anti-IgG, Light Chain Specific antibodies react with native primary antibodies used for detecting specific protein bands on Western blots (Figure 7 A). If diluted properly anti-light chain specific antibodies do not bind to the reduced and denatured IgG heavy chain band (50 kda) on blots (Figure 7 B, D, E and F). Therefore, by using anti-light chain specific antibodies, detection of antigens with molecular weights near 50 kda is not obscured by large amounts of reduced and denatured IgG heavy chains from primary antibodies used for immunoprecipitation (IP). Although the antibodies react strongly with native IgG light chains, some do not react as strongly with reduced and denatured light chains on blots (see for example 6A and D). Therefore, they are not recommended for sensitive and quantitative detection of reduced and denatured light chains. The antibodies have been thoroughly adsorbed to minimise cross-reactivity with immunoglobulins from many other species, which also may be present on blots. If the protein of interest has a reduced and denatured molecular weight near 25 kda, anti-igg, Fc fragment specific antibodies may be used to detect native IgG primary antibodies without binding to the 25 kda band of reduced and denatured IgG light chains on Western blots. Antibody Description IgG Fraction Monoclonal Mouse Anti-Goat IgG, Light Chain Specific*** (min X Hrs, Hu, Ms, Rb, Rat Ig) AffiniPure Goat Anti-Mouse IgG Light Chain Specific (min X Bov, Gt, Hrs, Hu, Rb, Rat, Shp Ig) IgG Fraction Monoclonal Mouse Anti-Rabbit IgG, Light Chain Specific (min X Bov, Gt, Ar Hms, Hrs, Hu, Ms, Rat, Shp Ig) AffiniPure Goat Anti-Rat IgG, Light Chain Specific (min X Bov, Gt, Hrs, Hu, Ms, Rb, Shp Ig) IgG Fraction Monoclonal Mouse Anti-Sheep IgG, Light Chain Specific (min X Bov, Hrs, Hu, Ms, Rb, Rat Ig) Figure 7. Detection of primary antibodies made in goat (against a 50 kda protein) by mouse anti-goat IgG, Light Chain (LC) specific (A). Heavy (50 kda) and light (25 kda) chains of reduced and SDS-denatured mouse IgG (B, C), goat IgG (D), rat IgG (E) and rabbit IgG (F) were separated by SDS-PAGE and detected on Western blots using HRP-goat anti-mouse IgG, LC specific (B), HRP-goat anti-mouse IgG (H+L) (C), HRPmouse anti-goat IgG, LC specific (D), HRP-goat anti-rat IgG, LC specific (E) and HRP-mouse anti-rabbit IgG, LC specific (F). No heavy chain band was detected even on lanes heavily overloaded with IgG when anti-igg, LC specific antibodies were used for detection (B, D, E and F). However, both heavy and light chain bands were detected with anti-igg (H+L) (C). The three left-hand lanes had reduced and SDS-denatured goat IgG (B, C and E) or mouse IgG (D and F) and the two right lanes in A had reduced and SDS-denatured mouse IgG, all of which served as background controls. The two middle lanes in A and the five right lanes in D demonstrate the relatively weak reactivity this antibody has with reduced and SDS-denatured light chains. *** Warning: BSA and dry milk may contain IgG which will be recognised by this antibody. Use of BSA or dry milk to block or dilute this antibody may increase background and/or reduce secondary antibody titre. 62 This antibody reacts primarily with kappa light chains. It is not suitable for detection of primary antibodies with lambda light chains.

63 Ordering Technical Anti-IgG, Light Chain Specific Antibodies for Western Blotting after IP Unconjugated Alexa Fluor 488 A=493, E=519 Cyanine Cy3 A=550, E=570 Alexa Fluor 594 A=591, E=614 Alexa Fluor 647 A=651, E=667 Biotin-SP (long spacer) Horseradish Peroxidase Alkaline Phosphatase Figure 7. Telephone: +44 (0) Fax: +44 (0)

64 Alexa Fluor 680 and Alexa Fluor 790 Conjugates for High Sensitivity Western Blots Antibodies conjugated with far-red and infrared-emitting dyes are more sensitive than dyes emitting visible light due to lower fluorescence quenching of the conjugates, higher extinction coefficients of the dyes and lower background autofluorescence (Figure 8). The increased brightness allows for a wider range of immunofluorescence detection and imaging modalities. Far-red and infrared dye conjugates can be used for higher sensitivity Western blots, quantitative Western blots, in-gel Western blots, microwestern arrays, incell Western arrays, on-cell Western arrays, tissue section imaging and other techniques that require the brightest dyes. Jackson ImmunoResearch offers the largest selection of Alexa Fluor 680 and Alexa Fluor 790 dyes conjugated with signal-enhancing primary antibodies, secondary antibodies, streptavidin and purified immunoglobulin controls for use in quantitative Western blots and other techniques requiring high sensitivity. The secondary antibodies are adsorbed for multiple labelling to minimise cross-reactions with others species and/or with other immunoglobulin classes. Figure 8. Double immunofluorescence staining on a Western blot using Alexa Fluor 680 far-red fluorescent dye (red) and Alexa Fluor 790 infrared fluorescent dye (green). Two micrograms of mouse IgG and two micrograms of goat IgG (background control) were reduced and denatured with β-mercaptoethanol and SDS. The heavy and light chains were separated by electrophoresis in SDS-PAGE, transferred to nitrocellulose and double labelled with a 1:100,000 dilution of Alexa Fluor 790-goat anti-mouse IgG, Fc subclass 1 specific (min X Hu, Bov, Rb Sr Prot), , to detect IgG heavy chains and a 1:100,000 dilution of Alexa Fluor 680-goat anti-mouse IgG, light chain specific (min X Bov, Gt, Hrs, Hu, Rb, Rat, Shp Ig), (red) to detect IgG light chains. Fluorescence was imaged in a LiCor Odyssey imager. Note the very faint bands in the goat IgG lane attesting to the extreme brightness of the dyes even at a dilution of 1:100,000. Figure *** Warning: BSA and dry milk may contain IgG which will be recognised by this antibody. Use of BSA or dry milk to block or dilute this antibody may increase background and/or reduce secondary antibody titre. ** Caution: See page 6 (min X... Sr Prot) before selecting an antibody adsorbed against closely related species. * ML = Multiple Labelling (see Multiple Labelling on pages 6-7 for an explanation). IgY is the original designation for the IgG-like protein found in both serum and egg yolk.