iba Strep-tag Strep-tag -Broschure Purification, Detection and Immobilization of Strep-tag proteins with Strep-Tactin

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1 iba Solutions For Life Sciences Strep-tag -Broschure Strep-tag Purification, Detection and Immobilization of Strep-tag proteins with The protein production system for highly pure proteins

2 THE STREP-TAG - TECHNOLOGY Strep-tag II Strep-Tactin Recombinant protein 1. Strep-tag II Only 8 amino acids Rapid one-step purification under physiological conditions High purity due to low tendency of Strep-Tactin for unspecific protein binding No tag removal necessary, N- and C-terminal fusion possible 2. Twin-Strep-tag Higher affinity for Strep-Tactin, same mild elution conditions More efficient capture of proteins from diluted cell culture supernatants Twin-Strep-tag 3. Strep-Tactin Purification of Strep-tag and Twin-Strep-tag proteins Detection of Strep-tag and Twin-Strep-tag proteins Strep-Tactin Strep-Tactin XT. NEW Strep-Tactin XT New developed variant for immobilization /assays pm affinity for Twin-Strep-tag tight binding Allows purification under denaturing conditions (6 M urea) His-STREPPER 5. His-STREPPER Adapter molecule which converts His-tag fusion proteins into Strep-tag fusion proteins No cloning required 6x-His-tag Strep-Tactin 6. Detection and Visualization Strep-Tactin and Strep-tag antibodies for detection Fluorescent conjugates HRP and AP conjugates Strep-tag -antibody 7. Immobilization Twin-Strep-tag binds to Strep-Tactin XT with an affinity in pm range - very low off-rate but mild elution still possible 2

3 CONTENT 1. Technologies Strep-tag II Twin-Strep-tag Strep-Tactin XT His-STREPPER Products Expression of (Twin-) Strep-tag proteins Purification of (Twin-) Strep-tag proteins...9 Strep-tag II Starter Kits... 9 Twin-Strep-tag Starter Kits WET-FRED Buffers and Reagents Resins MagStrep: Strep-Tactin coated Magnetic Beads Columns, cartridges, plate formats and adapters Detection of (Twin-) Strep-tag proteins Immobilization of (Twin-) Strep-tag proteins Strep-/ His-tag products for protein purification Ten Reasons to buy Strep-tag...backside Why we use Strep-tag! Why we admire the Strep -tag purification system: (i) generally good expression of recombinant proteins, (ii) simplicity of protein purification and very high purity of isolated proteins (the latter is very important for crystallographic projects!), (iii) robust system to generate, reliably purify, and biochemically analyze various mutant derivatives reproducibly. Prof. Dr. Erhard Bremer, Laboratory for Microbiology, Department of Biology, Philipps University Marburg, Germany. The Strep II tag appears to be an excellent candidate for affinity purification in general since it is a short tag that produces high purity material in good yields at a moderate cost. /... / we find that a combination of His-tag and StrepII tag allows rapid capture of the tagged protein or protein complex from crude extracts... Lichty et al. 2005, Protein Expression and Purification 1:

4 1. Technologies 1. TECHNOLOGIES 1.1 STREP-TAG For the use of this system we offer a large range of high quality products, such as Cloning/Expression Vectors for different hosts Various Starter Kits for newcomers Different purification resins and pre-packed columns Detection Systems with conjugates or a monoclonal antibody biotin streptavidin Facts about Strep-tag The Strep-tag / system is one of the most widely used affinity chromatography systems in protein purification, detection and immobilization. Advantages: Highly pure proteins due to specific binding Biological function of proteins is preserved The Strep-tag is a short peptide consisting of 8 amino acids. It was initially developed for the specific reversible binding to the biotin pocket of streptavidin. Streptavidin was later on engineered to obtain the highly selective, which binds the Strep-tag II with a nearly 100 times higher affinity compared to streptavidin. The Strep-tag II can be fused to the protein as either N- or C-terminal tag. Physiological buffers in combination with a wide range of additives can be used. The elution is performed competitively with desthiobiotin, an inexpensive, reversibly binding and stable analog of biotin. Strep-tag is a peptide that was engineered for the specific binding to the biotin binding pocket of Streptavidin. Strep-tag II the short 8-amino acid tag does not influence the protein. NH 2 -WSHPQFEK-COOH Strep-tag II Recombinant protein Strep-Tactin Strep-tag Protein Purification System The Strep-tag protein purification system provides the reliable one-step purification of proteins suitable for a broad range of applications, including e.g.: Structural and functional investigations Crystallization for determination of 3D structure Due to the mild conditions Strep-tag II recombinant proteins can also be used for: Assays involving protein-protein and protein-dna interactions Investigating ligand-receptor interactions under physiological conditions Separating living cells for re-culturing purposes Strep-tag is the method of choice for multiple protein classes: Metalloproteins / Enzymes Membrane proteins Sensitive protein complexes with multiple subunits And any other protein Recombinant protein with Strep-tag II bound to Strep-Tactin.

5 1.Technologies Strep-tag Principle Purification procedure under physiological conditions The purification of Strep-tag II fusion proteins is easy, straightforward and user-friendly. The complete procedure should be performed under physiological conditions at a ph > Binding Steps 1 + 2: The cell lysate is subjected to the column. Once the tagged protein has bound specifically to the host proteins are rapidly washed off due to addition of low amounts of physiological wash buffer (Buffer W). 2. Step 3: Then, bound Strep-tag II protein is gently eluted by addition of wash buffer supplemented with 2.5 mm desthiobiotin (Buffer E), which specifically competes for the biotin binding pocket. Since the buffer conditions during elution essentially remain unchanged, unspecific bound proteins (without Strep-tag II) will not be eluted and, thus, will not contaminate the protein of interest. Besides the specific binding of Strep-tag to, this is the second specificity conferring step of the purification procedure. Due to the competitive binding, only Strep-tag fusion protein will be eluted, yielding extremely high protein purity. 3. Elution Steps : In order to regenerate the column the yellow azo dye HABA (2- [ -hydroxy-benzeneazo] benzoic acid) is added as component of buffer R in excess to displace desthiobiotin from the binding pocket. Once HABA binds to the binding site, the color turns to red conveniently indicating the regeneration and activity status of the column.. Regeneration HABA (bound) Step 5: HABA can be removed simply by adding wash buffer. Once the red color disappeared the column can be re-used. resin can be regenerated and re-used 3 to 5 times without loss in performance. 5. columns regenerated with HABA (Sepharose, Superflow, Macroprep from left to right) 5

6 1. Technologies 1.2 TWIN-STREP-TAG Twin-Strep-tag Twin-Strep-tag : The high affinity, tandem Strep-tag for Protein Complex Purification and Protein:Protein Interaction Analysis Twin-Strep-tag : tandem arrangement of two Strep-tag II (total size of 30 aa) SA-WSHPQFEKGGGSGGGSGGSA WSHPQFEK The high affinity tag for Protein Complex Purification and Protein:Protein Interaction Analysis Based on the proprietary Strep-tag technology the Twin-Strep-tag a sequential arrangement of two Strep-tag II was developed. This tag enables the same mild and rapid purification as Strep-tag II, but it has a significantly increased affinity for and a better availability because of its larger size. Benefits of the Twin-Strep-tag Twin-Strep-tag has a neutral amino acid composition > 95 % pure protein after a single purification step High affinity leads to good protein yields Specific competitive elution with small amounts of (desthio-) biotin in the physiological wash buffer Tolerates elevated levels of additives and detergents Twin-Strep-tag is the tag of choice for highly diluted target proteins, e.g. from cell culture supernatants. Twin-Strep-tag is very well suited for the following applications Protein:Protein Interaction Analysis Protein Complex Isolation Purification of highly diluted proteins from e.g. cell culture supernatants Twin-Strep-tag should be used for proteins hampering the binding ability of Strep-tag II to. Due to its tandem arrangement of two Strep-tag II, the Twin-Strep-tag overcomes negative influences of the target protein. More information can be obtained from Schmidt et al., 2013 Development of the Twin-Strep-tag and its application for purification of recombinant proteins from cell culture supernatants Protein Expression and Purification; Volume 92, Issue 1 preys Twin-Strep-tag Protein:Protein Interaction Analysis The high specificity of the Twin-Strep/ interaction and low tendency of to bind proteins non-specifically makes the system very attractive for protein:protein interaction analysis. 3 bait 5 Strep-Tactin 6 protein 6 Superflow Features: Better availibility of the tag due to the sequential arrangement of two Strep-tag II High purity after one purification step High specificity - low tendency for false positive binding 6

7 1.Technologies Twin-Strep-tag and Strep-Tactin XT for immobilization and assays The development of Strep-Tactin XT (see next chapter) extends the use of Twin-Strep-tag to the field of assay applications. Features: pm affinity of Twin-Strep-tag to Strep-Tactin XT stable binding Reversibility of Twin-Strep/Strep-Tactin XT binding High specificity- low background Stable under physiological conditions Allows usage of detergents and additives As a peptide tag Twin-Strep-tag is especially used in purification of protein complexes, given that its size has nearly no influence on the complex formation in vivo compared to protein tags, like MBP or GST. 1.3 STREP-TACTIN XT Strep-tag conquers the world of assay implementation In order to overcome certain limitations of the Strep-tag technology with respect to binding stability, in the field of assay usage the binding affinity of Strep-tag II to had to be improved. Therefore, Strep-Tactin XT was engineered. Strep-Tactin XT has a binding affinity in double digit pm range for Twin-Strep-tag and in nm range for Strep-tag II. This, improvement in binding stability especially for Twin-Strep-tag makes the Strep-tag technology amenable to assay applications. Strep-Tactin XT Advantages of Strep-Tactin XT: Allows the immobilization in very stable ranges (T 1/2 = 13 h) The interaction with (Twin-) Strep-tag is still reversible Improves the performance of the Strep-tag technology in assay s Purification even under denaturing conditions (6 M urea) possible Strep-Tactin XT Edition: All products conjugated with the new high affinity Strep-Tactin XT are labelled with XT in the product name. When to use which TAG with which STREP-TACTIN RESIN? Strep-Tactin XT Application and Conditions Strep-tag II Twin-Strep-tag Strep-tag II Twin-Strep-tag Concentrated (cytosolic) Purification Diluted (secretion) Batch (magnetic beads) 8 Denatured (6M urea) 8 8 Detection (Western, ELISA) Assay/immobil. (MTP, chips) 8 7

8 1. Technologies Strep-tag II His-STREPPER Tris-NTA 1. His-STREPPER Strep your His-tag without cloning His-STREPPER adapter molecule converts a 6xHis-tag fusion protein into a Strep-tag II carrying protein. The molecule comprising Strep-tag II (SA-WSHPQFEK) conjugated with a nickel charged. It tightly binds to 6xHis-tag and thereby converts a 6xHis-tag fusion protein to a Strep-tag II fusion protein without the need for cloning. It gives access to the advantages of the Strep-tag purification system, namely the high purity of the isolated proteins, without a time-consuming cloning process to His-tag users. (More information on page 19) His-STREPPER conveys the benefits of the Strep-tag Technology to 6xHistidine fusion proteins. Features: Adapter molecule for fast and easy conversion of 6xHistidine-tag fusion protein to Strep-tag II fusion proteins Transfer all Strep-tag II advantages (pure & functional proteins) to 6xHis-tag proteins Cost- and time-effective His-STREPPER purifies 6xHis fusion proteins to higher purity Purification results for GFP-His using different purification protocols. GFP-His was either purified using the His-STREPPER and in PBS ph 8 buffer or using Ni-NTA under the same physiological or non-physiological conditions. His- STREPPER:Strep-Tactin provides better results than His-tag:Ni-NTA. Crude lysate + Ni-NTA-Superflow + His-STREPPER + Strep-Tactin-Superflow Purity = % Purity = 99% 2. PRODUCTS 2.1 EXPRESSION ColEl ori tet A pasg-iba f1 ori Combinatorial Sites For expression of Strep-tag II and Twin-Strep-tag fusion proteins IBA offers a variety of expression vectors (StarGate Cloning System) For E. coli, mammalia, insect cells and yeast Strep-tag, Twin-Strep-tag, His-tag, GST-tag and FLAG-tag vectors tet repressor AmpR More information can be found on our homepage 8

9 Furthermore the MEXi (Mammalian Expression IBA) system was developed to provide an optimized system for expression of Twin-/ Strep-tag II fusion proteins in mammalian HEK293 cells. It comes along with a HEK293E cell line (MEXi 293E), a transfection (MEXi -TM) and cultivation (MEXi -CM) medium and an optimized vector system (pdsg-iba). MEXi Mammalian Expression IBA system: High protein yields Easy handling Optimized components Cost-effective 2.2 PURIFICATION STREP-TAG II STARTER KITS Attractive offers for newcomers and experts are our Strep-tag II Starter Kits containing all essential reagents required for expression in E. coli, purification and detection of Strep-tag II proteins. All Strep-tag II Starter Kits include: Control plasmid with a 15 kd protein insert Anhydrotetracycline for induction of expression Fractionation buffer for the preparation of a periplasmic extract Wash buffer for column chromatography or for the preparation of a cytoplasmic extract Elution buffer for displacing the Strep-tag II protein from the column Column regeneration buffer (with HABA) horse radish peroxidase (HRP) conjugate for Western blot detection. Strep-tag-Starter Kit with one purification column In addition the different Starter Kits are equipped with different columns or cartridges to enable the evaluation of the optimal resin for a particular protein of interest: Strep-tag Starter Kit Contains one ready-to-use column with Sepharose. Cat. no Strep-tag Starter Kit 3C Includes 3 different columns with immobilized to Sepharose, MacroPrep and Superflow, respectively, allowing the evaluation of the optimal resin for your particular protein of interest. Cat. no Strep-well HT 50 Purification Starter Kit The perfect solution for automated, high-throughput purification of Strep-tag proteins. Allows simultaneous purification of up to 96 different Strep-tag proteins and yields in up to 200 µg highly pure Strep-tag protein per well. Cat.no:

10 TWIN-STREP-TAG - STARTER KIT Further methods for PPI analysis are our tandem affinity purification kits: One-TAP - With Twin-Strep-tag only Two-TAP - Using two affinity tags (Twin-Strep-tag/FLAG-tag For both is a Cloning Kit, for either mammalian cells or E. coli available For Twin-Strep-tag newcomers we provide kits for cloning and for purification respectively. StarGate Twin-Strep Cloning Kit, E. coli ( ) StarGate Twin-Strep Cloning Kit, mammalia ( ) Twin-Strep Basic Purification Kit ( ) The Cloning Kit provides all necessary components for cloning a certain gene of interest (GOI) into Twin-Strep-tag vectors and the Basic Purification Kit consists of all components required for a first purification trial. Note, that the components of the Basic Purification Kit are adjusted to the protein:protein interaction procedure where a single-use of columns is recommended. For protein:protein interaction analysis in combination with Twin-Strep-tag further methods are described. Detailed information about our protein:protein interaction technologies and products are available on our homepage ( WET FRED Components of WET FRED (Twin-) Strep-tag purification from cell culture supernatant In mammalian and insect cell expression recombinant proteins are often secreted in the cell culture medium. Thus, the protein is present in large volumes before the purification step. To apply this large volume onto a gravity flow column with a certain flow the WET FRED applicator was developed. This device facilitates the transfer of large volumes to a gravity flow column for purification of the recombinant target protein fused with (Twin-) Strep-tag. It works simply by hydrostatic pressure (siphon principle). Due to its small size and flexibility it is easy to handle at the bench, in the cold room or in the fridge. Columns cannot run dry and need no supervision. Furthermore, no sophisticated software is necessary facilitating set up and use. height to be adjusted for flow regulation Cat.no for 1ml columns and for 5-10ml columns Experimental set-up 10

11 BUFFERS AND REAGENTS Product Contents Cat.no Avidin (high grade for PPI applications) Anhydrotetracycline (inducer for tet promotor) BioLock biotin blocking solution Strep-tag protein purification buffer set 15 mg 50 mg 25 mg 50 mg 50 ml 250 ml D-Desthiobiotin 1 g, 5 g Elution Buffer with D-Desthiobiotin (10x Buffer E) Strep-tag regeneration buffer with HABA (10x Buffer R) 100 ml 10x Buffer W 25 ml 10x Buffer E 100 ml 10x Buffer R ml ml Strep-tag washing buffer (10x Buffer W) 100 ml Strep-tag II Peptide 1.8 mg Biotin Elution Buffer (10x Buffer BE) 25 ml Biotin Elution Buffer (5x Buffer BX) for MacStrep type3 XT 50 ml columns regenerated with HABA (see also Strep-tag purification cycle): Sepharose, Superflow and MacroPrep (left to right). Since MacroPrep is not as transparent as Sepharose or Superflow, the color shift to red is less visible. BioLock biotin blocking solution Culture media often contain significant amounts of biotin. This is especially the case for mammalian or insect cell culture media. When proteins from biotin containing extracts or media are intended to be purified via chromatography, biotin must be masked by the addition of avidin prior to the application onto the column. BioLock biotin blocking solution allows this masking of biotin (and biotinylated proteins) in a very fast and convenient way. Activity: >70U/ml; add at least 1U of BioLock solution per µg of biotin. Cat.no d-biotin Biotin Elution Buffer BX The new Strep-TactinXT variant requires a more competitive elution buffer due to the higher affinity of Strep-Tactin XT for Strep-tag II and especially Twin-Strep-tag. Biotin Elution Buffer BX has a higher biotin concentration to enable an efficient elution from e.g. MagStrep type3 XT beads. Cat.no

12 STREP-TACTIN RESINS Which Resin to use? Test our different resins to evaluate the best one for the purification of your protein of interest. The Strep-tag Starter Kit 3C ( ) consits of 1 ml gravity flow columns of -Sepharose, -Superflow and -MacroPrep, respectively. Different types of resins are provided Several resin versions are available which differ in their properties and suitability for applications. Sepharose Superflow, Superflow High Capacity (HC) MacroPrep MagStrep Beads ( XT coated magnetic Beads) is preferentially used for gravity flow chromatography. can also be used for low pressure, FPLC and HPLC applications. In addition, Superflow is especially suited for increased flow rates and for the purification of large protein complexes (see: for batch purification only. MAGSTREP:STREP-TACTIN COATED MAGNETIC BEADS MagStrep (Strep-Tactin XT coated Magnetic Beads) type3 XT enable the fast purification of Strep-tag fusion proteins in batch formate. In addition, it allows purification from small volumes. Features: High binding capacity (1-3 nmol/µl beads, corresponding to µg of a 30 kda protein) Very low non-specific protein binding due to improved coating Flexible elution conditions, under denaturing conditions by boiling in SDS gel loading buffer or under native conditions with biotin High affinity for Twin-/Strep-tag II due to new Strep-Tactin XT Crude bacterial extract before purification MagStrep type3 XT beads: Cat.no Magnetic separator: Cat.no Biotin Elution Buffer (5x Buffer BX): Cat.no Native purification using buffer BX for elution Denaturing purification using sample buffer and boiling for elution GFP-Strep-tag GFP-Strep-tag Strep-Tactin XT GFP-Strep-tag Purity > 95% Purity > 95% Fig: Purification of GFP-Strep-tag fusion protein from crude bacterielle extract using MagStrep type3 XT beads. Due to specific binding properties of the beads even the elution by boiling leads to highly pure proteins. (Protein purification analysis was performed with an Agilent Bioanalyzer 2100 system instead of SDS-PAGE) 12

13 Different Resin Specifications Sepharose Superflow Superflow high capacity MacroPrep MagStrep Magnetic Beads Gravity flow column Yes Yes Yes Yes No Batch purification For Twin-Strep-tag only For Twin-Strep-tag only For Twin-Strep-tag only For Twin-Strep-tag only FPLC No Yes Yes Yes No HPLC No Yes Yes Yes No Binding capacity nmol/ml nmol/ml nmol/ml nmol/ml Type3: 1-3 nmol/µl beads Support Sepharose FF Superflow 6 Superflow 6 MacroPrep 50 / Bead structure agarose 6 % agarose, crosslinked Bead size µm µm spherical 6 % agarose, crosslinked µm spherical polymethacrylate Yes* 50 µm Type3: 25 µm Exclusion limit 3 x x x x 10 6 / Recommended linear flow rate up to 30 cm/h up to 300 cm/h up to 300 cm/h up to 300 cm/h / ph stability n.d. Max. pressure Gravity flow 9.6 bar (cartridge cover 3 bar only) 9.6 bar (cartridge cover 3 bar only) 70 bar (cartridge cover 3 bar only) * For Strep-tag II only recommended if expression rate >1 mg/ml otherwise use Twin-Strep-tag / COLUMNS, CARTRIDGES, PLATE FORMATS AND ADAPTERS No Description Application Volume applied ml gravity flow columns (5 columns) Sepharose Superflow Superflow HC MacroPrep Gravity flow ml ml gravity flow column Gravity flow ml ml gravity flow column Gravity flow ml ml gravity flow column Gravity flow ml ml cartrigde FPLC/HPLC ml ml cartrigde FPLC/HPLC ml Spin Columns (for 150 µg protein each) 6 Strep-Well HT 50 plates (10 x 96well plates, for 100 µg protein/well) Spinning High throughput/ Vacuum based Up to 500 µl Kit: , µl 1 ml Kit: Adapters for H-PR cartrigdes Syringe adapter set (Luer-lock) Cat. No /16 inch adapter set for peristaltic pump tubing Cat. No M6 adapter set for GE Healthcare FPLC other than ÄKTA TM Cat. No /-28 adapter set for FPLC other than GE Healthcare Cat. No Coupling adapter set for connecting up to three cartridges Cat. No

14 2.3 TWIN-/STREP-TAG PROTEIN DETECTION SYSTEM The following monoclonal antibodies against Strep-tag are available: StrepMAB-Classic, unconjugated StrepMAB-Classic HRP conjugate (HRP, horseradish peroxidase) StrepMAB-Immo - the high affinity antibody for capturing Strep-tag proteins on solid surfaces Direct detection of recombinant Strep-tag II GFP in a Western blot using HRP conjugate % of Max APC-A Zymosan_Unstained.fcs 21.1 Zymosan_C MAB immo O65.fcs 3.6 Zymosan_Clec7 MAB immo O65.fcs 2581 FACS analysis of Zymosan, stained with StrepMAB Immo Oyster 65 against Clec7-One-STrEP-tag 1:100, 20 min ice Kindly provided by: Dr. K. Neumann, III Med. Klinik, TUM München Immunofluorescence of type-i transmembrane protein (extracellular domain N-terminal Streptagged) stained with Chromeo 56 1:100 in insect cells. Fixation: % Formaldehyd in PBS, 20minuten RTKindly provided by: Dr. Michael Krahn,, Abteilung Stammzellbiologie, GZMB Göttingen The Strep-tag protein detection system supports a broad variety of assays, including: Western blot and ELISA procedures (colony blot, dot blot) Screening for positive expression clones Monitoring expression levels and stability of Strep-tag proteins Immunocytochemistry and immunohistochemistry Protein localization and targeting studies Detection of Strep-tag fusion proteins in Western blots/elisa For direct detection of Strep-tag fusion proteins in Western blots via chemiluminescence reaction IBA offers: HRP conjugate (HRP, horseradish peroxidase) StrepMAB-Classic HRP conjugate. (no secondary antibody is needed!) For direct detection of Strep-tag fusion proteins in Western blots via chromogenic reaction use AP conjugate (AP, alkaline phosphatase) HRP conjugate (HRP, horseradish peroxidase) For protein detection in ELISA use StrepMAB-Classic, unconjugated HRP conjugate AP conjugate Detection of Strep-tag fusion proteins in Immunofluorescence/FACS For the detection of Strep-tag fusion proteins in Immunofluorescense and FACS use StrepMAB-Classic, unconjugated StrepMAB-Immo, unconjugated Or directly conjugated, StrepMAB-Classic StrepMAB-Immo labeled with Chromeo 88, Chromeo 56 or Oyster 65 respectively. 1

15 Two different detection systems are available to detect Strep-tag II as well as Twin-Strep-tag at the N-terminus, C-terminus: a) Monoclonal antibodies (mab), conjugated and unconjugated b) conjugates Strep-tag -antibody Product overview: Detection System Description Features Secondary antibody required (cat. no ) Western blot, chromogenic detection Western blot, chemiluminescent detection (ECL) Immunofluorescence, ELISA, FACS* Detects also biotinylated proteins a) Monoclonal antibodies b) conjugates StrepMAB- Classic Strep-tag IIand Twin-Strep-tag - specific monoclonal antibody, unlabeled - Highly selective - Low background Yes, secondary anti-mouse IgG, HRP-conjugated, required Suited, but not recommended Not recommended Cat. no (100µg) Available as complete kit with all reagents required StrepMAB- Classic HRP conjugate Strep-tag IIand Twin-Strep-tag - specific monoclonal antibody, labeled with horse radish peroxidase - Highly selective - Low background No, direct detection via HRP HRP conjugate protein, labeled with horse radish peroxidase - Sensitive - Fast detection protocols No, direct detection via HRP AP conjugate protein, labeled with alkaline phosphtase - Very sensitive - Fast detection protocols No, direct detection via AP Recommended Recommended Recommended Recommended Recommended Not determined Recommended For ELISA only For ELISA only For ELISA only (75µg for25-30 Western Blots) (0.5 ml) (0.5 ml) The Strep-tag Protein Ladder is designed for accurate MW determination on Coomassie Blue stained gels and as positive control on Western blots. As each protein contains the Strep-tag II sequence which is detected by our conjugates or Strep-tag specific antibodies, the ladder can also be used for MW determinations on Western blots and serves as a positive control for the various detection systems. Cat.No Fluorescent conjugates: Labelling StrepMAB-Classic StrepMAB-Immo Chromeo 88 Chromeo 56 Oyster µg amounts Available as 50 µg and 100 µg 50 µg amounts µg amounts mab and Fab for different species available: StrepMAB Classic and StrepMAB Immo are available as full length antibodies and Fab for human, mouse, rat or rabbit. Request at info@iba-lifesciences.com. 15

16 2. TWIN-/STREP-TAG -PROTEIN IMMOBILIZATION Immobilization of proteins can be useful for the following applications: Antibody or serum screening Diagnostic assays Expression cloning Protein interaction studies Screening of engineered enzymes Drug screening Strep coated microplates and 8 well strips Related IBA products Strep-Tactin XT coated microplates Via Strep-Tactin XT Antibody-free option for immobilization of Strep-tag proteins The ready-to-use Strep-Tactin XT coated microplates provide the power of the Strep-tag system in a solid-phase, multi-well format for convenient assays and high-throughput screenings of biomolecules tagged with Twin-Strep-tag. The combination of Strep-Tactin XT with the Twin-Strep-tag is highly stable with a T 1/2 of 13 hours and an affinity in pm range. The strips of 8 wells are supplied in sets of 12 resulting in a 96-well plate. These 96-well plate configuration is compatible with standard multichannel pipettes, automated plate washers and plate readers. The biomolecules are presented to interacting partners in a uniform manner which results in reliable and reproducible assay formats. In addition a beneficial feature of Strep-Tactin XT coated microplates is the possibility of regeneration of the plate. Features: Oriented binding of recombinant proteins with N-terminal or C-terminal Twin-Strep-tag Minimal non-specific binding Minimal coefficients of variation High affinity and stability of Strep-Tactin XT with Twin-Strep-tag Cost-effective Reversibility along with high affinity Capture of functional target proteins on Strep-Tactin XT coated microplates Using the MTP assay the binding capacity of Strep-Tactin XT was examined and compared to Strep-Tactin. For this purpose BAP was fused with Streptag II and applied in different amounts onto the Strep-Tactin/Strep-Tactin XT coated microplates. After washing the remaining amounts of BAP were measured. Resulting in almost 100 % recovery* of BAP-Strep-tag II on Strep-Tactin XT compared to 8 % recovery on Strep-Tactin. chromogenic color substrate AP reaction BAP-Strep-tag II *red area 16

17 Via StrepMAB-Immo StrepMAB-Immo antibody StrepMAB-Immo antibody is the reagent of choice for efficient immobilization of Strep-tag II proteins on solid phases. StrepMAB- Immo is a murine, high-affinity Strep-tag II specific monoclonal antibody which is especially suited for stable, mild and oriented immobilization of Strep-tag II fusion proteins. To realize this, the antibody can be coated on e.g. microplates, columns, Biacore CM5 sensor chips or other biochips. The nearly irreversible binding is achieved for fusion proteins carrying a C- or N- terminal Strep-tag. But the Strep-tag must be extended at the N-terminus by a SerAla linker (recombinant protein- SA-WSHPQFEK or SA-WSHPQFEK-recombinant protein). StrepMAB-Immo, high-affinity Strep-tag II (Twin-Strep-tag ) specific monoclonal antibody for capturing Strep-tag fusion proteins on solid phases StrepMAB-Immo coated microplates This antibody-based highly efficient immobilization of Strep-tag proteins achieves a high wash stability. StrepMAB-Immo coated microplates can be used for efficient, mild and oriented immobilization of SerAla-Strep-tag II fusion proteins for ELISA or other assays used for protein analysis. Also small amounts of such proteins are bound with high efficiency to the microplate and will not elute during the assay due to nearly irreversible binding activity of StrepMAB-Immo. Features: Ready-to-use 96-well plates for your own protein assay Efficient immobilization of minute amounts of SerAla-Strep-tag II fusion proteins saves your starting material High wash stability during the assay Related IBA products StrepMAB-Immo high affinity antibody StrepMAB-Immo coated microplates Comprehensive IBA portfolio for protein purification and analysis product selection guide Strep-Tactin XT Application and Conditions Strep-tag II Twin-Strep-tag Strep-tag II Twin-Strep-tag Concentrated (cytosolic) Purification Diluted (secretion) Batch (magnetic beads) 8 Denatured (6M urea) 8 8 Detection (Western, ELISA) Assay/immobil. (MTP, chips) 8 17

18 2.5 STREP-/ HIS-TAG PRODUCTS FOR PROTEIN PURIFICATION IBA provides two different Ni-NTA supports: Ni-NTA Sepharose for purification of 6xHistidine- tag proteins by gravity flow. Cat.No Ni-NTA Superflow for purification of 6xHistidine-tag proteins by gravity flow and FPLC Cat.No Purification of highly pure full-length proteins Protein expression is a complex topic with many variables. Therefore, it is e.g. hard to predict whether a recombinant protein is expressed soluble or forms inclusion bodies or is partially degraded. To be prepared for the most common difficulties the attachment of two different tags at each terminus of the recombinant protein provides the flexibility to obtain a highly pure and homogenous protein preparation. Important reasons for two different affinity tags on one protein are Purification of 100% full length proteins Highest purification grades Using denaturing or physiological purification conditions A smart double-tag pair is the combination of Strep-tag and 6xHistidine-tag. Generally, it is recommended to attach one tag to the N-terminus and the other to the C-terminus. Strep/6xHistidine-tag Starter Kit with Superflow high capacity This unique Starter Kit contains all reagents essential for the native purification of a double-tag protein with Strep-tag and 6xHistidine-tag. The first purification is performed on a Ni-NTA Superflow cartridge while the second purification uses a Superflow high capacity cartridge, selecting for 6xHistidine-tag and Strep-tag, respectively. Cat.no The double-tag protein purification process Ni-NTA cartridge Ni-NTA Possible variants in case of proteolysis Strep-tag Protein of interest cartridge 6xHistidine -tag Ni-NTA Resins 6xHistidine-tag is an optimal partner for Strep-tag in double tag proteins. The combination enables the rapid control of full length expression and increases protein purity due to two independent purification methods. IBA provides all necessary products needed for the double tag purification. 18

19 Gravity flow StrepMAB-Classic MacroPrep Column An antibody-based option for Strep-tag protein purification This antibody-based purification column with StrepMAB-Classic immobilized to MacroPrep provides an alternative for purification of Strep-tag proteins. Using this column as second purification step after can increase the purity to > 95 % while only one tag the Strep-tag II is used. In protein:protein interaction analysis, e.g. this column is used in the so-called One-TAP system enabling a tandem affinity purification (TAP) with two different immobilized receptors and one tag (Strep-tag II) only. Cat.no (1 ml); (5 x 0.2 ml) Strep /6xHistidine Starter Kit His-STREPPER The His-STREPPER adapter molecule converts a 6xHis-tag fusion protein to a Strep-tagged protein without cloning. Thereby it enables purification of initially His-tagged proteins with the Strep-tag II:Strep-Tactin system to improve purity of the His-tag fusion protein. Cat.no xHis-tag fusion protein His-STREPPER Strep-Tactin 6x His-tag/ Ni-NTA His-STREPPER/ Strep-Tactin Target protein Fig.: Use of His-STREPPER improves purity of 6xHis-tag fusion protein from 30% to 80% A 6xHis-tag fusion protein from crude bacterial cell extract was applied to Ni-NTA resin. After washing, the bound protein was eluted using Ni-NTA Elution Buffer (Qiagen, lane 2). Protein purity of the 6xHis-tag fusion protein (arrow) after elution was 30% only. To improve the purity of the eluted protein a further purification step was necessary. Therefore the elution fraction was dialysed to PBS ph 8.0 and the His- STREPPER adapter molecule was added to convert the His-tag into a Strep-tag fusion protein. The elution fraction was applied to and the converted protein was eluted with PBS ph 8.0; 2.5 mm desthiobiotin (lane 3). The addition of His-STREPPER and purification via leads in this protein sample to an increase of the protein purity from 30% to 80 %. (Protein purification analysis was performed with an Agilent Bioanalyzer 2100 system instead of SDS-PAGE.). 19

20 10 reasons to buy Strep-tag The complete system for protein expression, purification, detection, immobilization, assay and interaction 1. Highly pure proteins (> 95%) 2. Functional proteins due to physiological conditions 3. Fast one-step purification requiring a low washing volume only - due to low non-specific binding. Variable buffer conditions; high salts, detergents, metal ions, chelators or reducing agents can be used 5. Low background specific Strep-tag / interaction and competitive elution with desthiobiotin 6. Economic: re-usable, robust purification resins regeneration is color controlled 7. Preserves protein complex integrity - mild elution conditions, low washing volumes 8. Efficient immobilization via Strep-Tactin XT (reversible binding) or StrepMAB-Immo (non-reversible) 9. No tag removal required due to neutral pi. It does not influence protein folding and function 10. Universal Detection System for Western Blot, ELISA, Immunofluorescence, FACS and more Version PI