Avidin-Biotin Technical Handbook

Thermo Scientific Avidin-Biotin Technical Handbook Table of Contents EZ-Link Biotinylation Reagents Introduction Biotin-Labeling Reagent Selection Guides 1 2-3 Selection Guide 1 – Reagent Selection by Application Selection Guide 2 – Reagent Selection by Functional Group 3 Amine-Reactive Biotinylation Reagents 4-11 Introduction Amine-Reactive Biotinylation Kits Amine-Reactive Biotinylation Reagents 4 5 6-11 2 Sulfhydryl-Reactive Biotinylation Reagents 12-15 Carboxyl-Reactive Biotinylation Reagents 16-17 Carbohydrate/Aldehyde-Reactive Biotinylation Reagents 18-19 Photo-Reactive Biotinylation Reagents 20-21 Specialty Biotinylation Reagents 22-25 Avidin-Biotin-Based Kits 26 Protein Labeling – Solid Phase Biotinylation Kits 27 Protein Extraction Cell Surface Protein Isolation Kit Far-Western Blotting Pull-down Kit for Biotinylated Proteins Avidin-Biotin Binding Introduction Immobilized Avidin Products Immobilized Streptavidin Products Immobilized NeutrAvidin Products Immobilized Monomeric Avidin and Kit Immobilized Iminobiotin Thermo Scientific MagnaBind Beads NeutrAvidin Coated Polystyrene Plates NeutrAvidin High binding Capacity Coated Plates Streptavidin Coated Polystyrene Plates Streptavidin HBC Coated Plates 28-29 28 29 29 30-38 30 31 32 32 33 33 34 35 36 37 38 Protein Immunodetection 39-43 NeutrAvidin Products Streptavidin Products Avidin Products ABC Staining Kits Biotin Conjugates Example Protocols for Biotinylation 39 40 41 42 43 44-46 Troubleshooting Guide for Biotinylation with NHS-esters Biotinylating Cell Surface Proteins One-Step Biotinylation and Dialysis in a Thermo Scientific Slide-A-Lyzer Cassette Example Protocols for Affinity Purification Based on Avidin-Biotin Binding 45 45 46 47-48 Introduction Affinity Purification of Biotinylated Molecules Afffinity Purification Using a Biotinylated Antibody Immunoprecipitation Using a Biotinylated Antibody 47 47 48 48 Thermo Scientific EZ-Link Biotinylation Reagents S H • Carbohydrate residues containing cis-diols can be oxidized to create active aldehydes (–CHO). Antigen-binding site Light Chains VL VL VH CL CL S-S S- S CH1 CH1 Hinge Region NH2 Papain S-S S-S Carbohydrate VH Fab (Fab')2 S-S S- NH2 S While NHS-esters of biotin are the most frequently used biotinylation reagents, they are not necessarily the best for a particular application. If only a portion of the primary amines on a protein are reacted, reaction with NHS-esters of biotin will result in a random distribution • Sulfhydryl groups (–SH) can be generated by reducing disulide bonds in the hinge region. S S- The most frequently used biotinylation reagents, N-hydroxysuccinimide (NHS) esters and N-hydroxysulfosuccinimide (sulfoNHS) esters, react with primary amines. The functional groups available on the surface of the protein to be biotinylated may not be known. However, with most proteins, it is safe to assume that primary amines are available and accessible for biotinylation. The likelihood that primary amines are present increases as molecular weight increases. For example, BSA contains 59 primary amines and 30-35 of these are on the surface and can be reacted with NHS-esters. • Primary amine groups (–NH2) are found on lysine side chains and at the amino terminus of each polypeptide chain. S S- The valeric acid side chain of the biotin molecule can be derivatized to incorporate various reactive groups that are used to attach biotin to other molecules. Using these reactive groups, biotin can be easily attached to most proteins and other molecules. Biotinylation reagents are available for targeting a variety of functional groups, including primary amines, sulfhydryls, carbohydrates and carboxyls. Photo-reactive biotin compounds that react nonspecifically upon photoactivation are also available. This variety of functional group specificities is extremely useful, allowing the choice of a biotinylation reagent that does not inactivate the target macromolecule. Several cleavable or reversible biotinylation reagents are also available and allow specific elution of the biotinylated molecule from biotin-binding proteins. A complete selection guide and detailed instructions for each reagent is available on the “Products” section of our web site. Understanding the functional groups available on an antibody is the key to determining a strategy for modification. S Biotin MW 244.3 S NH HO S- O NH S- O H S Valeric Acid Side Chain Antibodies are biotinylated more often than any other class of proteins and it is advantageous to biotinylate in a manner that will maintain immunological reactivity. Thermo Scientific Sulfo-NHSLC-Biotin is the number one choice for labeling both monoclonal and polyclonal antibodies because it is the simplest and often most effective method. The fast and reliable procedure has been optimized for antibody modification. If the antibody contains a lysine-rich antigen-binding site, amine-reactive reagents may inhibit antigen binding. One solution is to use biotin derivatives that react with sulfhydryl groups. By reducing the immunoglobulin under mild conditions, biotinylation can be isolated to free sulfhydryls generated from the hinge region. Another solution is to use a biotin derivative such as Biocytin-Hydrazide that reacts with aldehydes. Aldehydes can be generated on antibodies and other glycoproteins by oxidation of carbohydrates with periodate. Because carbohydrate is found selectively on the Fc portion of antibodies, biotin labeling is restricted from occurring near the antigen-binding site. This site-specific labeling method provides an antibody conjugate with the highest possible specific activity that is particularly important when antibody supply is limited and making the best possible use of the antibody is desired. This method is particularly useful for labeling polyclonal antibodies, which are heavily glycosylated. S- Biotin, a 244 dalton vitamin found in tiny amounts in all living cells, binds with high affinity to avidin, streptavidin and Thermo Scientific NeutrAvidin Biotin-Binding Protein. Since biotin is a relatively small molecule, it can be conjugated to many proteins without significantly altering their biological activity. A protein can be reacted with several molecules of biotin that, in turn, can each bind a molecule of avidin. This greatly increases the sensitivity of many assay procedures. of biotin on the surface of the protein. If a particular primary amine is critical to the biological activity of the protein, modification of this critical amine may result in the loss of its biological activity. Depending on the extent of biotinylation, complete loss of activity may occur. SS The highly specific interaction of avidin with biotin (vitamin H) can be a useful tool in designing nonradioactive purification and detection systems. The extraordinary affinity of avidin for biotin (Ka = 1015 M-1) is the strongest known non-covalent interaction of a protein and ligand and allows biotin-containing molecules in a complex mixture to be discretely bound with avidin conjugates. Our extensive line of biotinylation reagents, conjugates and affinity supports exploits this unique interaction. Some applications in which the avidin-biotin interaction has been used include ELISA; immunohistochemical staining; Western, Northern and Southern blotting; immunoprecipitation; cell-surface labeling; affinity purification; and fluorescence-activated cell sorting (FACS). Pepsin CH2 S S S CH 2 S Carbohydrate CH3 S S S CH 3 S Heavy Chains FC NH2 NH2 Amines on lysine residues Sulfhydryls created when antibody is reduced To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor. 1 Thermo Scientific Pierce Biotin-Labeling Reagent Selection Guides These selection guides are designed to help you quickly choose the most appropriate biotinylation reagent to use for an application. Selection Guide 1 focuses on the purpose/application of biotin labeling and is organized by the type of molecule that is to be labeled. It includes only the most common biotinylation reagent choices. Selection Guide 2 focuses on reaction chemistries and is organized according to the functional groups present on the molecule to be labeled. For a more complete selection guide and for detailed product instructions, visit the “Products” section of our web site. Selection Guide 1 Biotinylation Target Application Thermo Scientific Pierce Reagent(s) Features/Benefits Antibody ELISA or blotting (to detect with streptavidin-HRP) or affinity-purifying antigen (after immobilizing on streptavidin column) Sulfo-NHS-LC-Biotin (Product # 21335, Kit Product # 21435, 21935) Amine-reactive (lysines). Sure to work well for nearly any antibody. Very simple and direct. NHS-PEG4-Biotin (Product # 21329, 21330, 21362) Amine-reactive (lysines). PEG spacer arm increases solubility of biotinylated molecule. Purified protein Purified peptide ELISA or blotting (to detect with streptavidin-HRP) or affinity-purifying receptor or antibody (after immobilizing on streptavidin column) ELISA or blotting (to detect with streptavidin-HRP) or affinity-purifying receptor or antibody (after immobilizing on streptavidin column) Cell surface proteins Primarily for affinitypurifying or removing cell surface receptor ligands (after immobilizing on streptavidin column) DNA/RNA or oligo-nucleotides 2 Capture or detection of oligonucleotides in ELISA-type applications or affinity purification (Capture in streptavidin-coated plates or detect protein-bound oligo with streptavidin-HRP) Biotin-PEG4-Hydrazide Carbohydrate-reactive. Primarily for polyclonal antibodies (purified from serum), (Product # 21360), plus which usually have required carbohydrate side chains. Sodium meta-Periodate (Product # 20504) Sulfo-NHS-LC-Biotin (Product # 21335, Kit Product # 21435, 21935, 21362) Amine-reactive (lysines). Obvious choice for sizeable proteins. Slight risk of blocking epitopes or binding sites necessary for other purposes. NHS-PEG4-Biotin (Product # 21329, 21330) Amine-reactive (lysines). PEG spacer arm increases solubility of biotinylated molecule. Sulfo-NHS-SS-Biotin (Product # 21331) Amine-reactive (lysines). Cleavable disulfide spacer allows recovery of biotinylated protein from immobilized streptavidin. Maleimide-PEG2-Biotin (Product # 21901, 21902) Sulfhydryl-reactive. Use only when there is a reason to target sulfhydryls rather than amines. Biotin-PEG4-Hydrazide (Product # 21360), plus Sodium Periodate (Product # 20504) Carbohydrate-reactive. Good choice if the protein is known to be adequately glycosylated and the carbohydrate is not important for downstream applications. Sulfo-NHS-LC-Biotin (Product # 21335, Kit Product # 21435, 21935) Amine-reactive. Choose if N-terminus or lysines are not required for binding reactions. May be difficult to remove unreacted reagent. NHS-PEG4-Biotin (Product # 21329, 21330) Amine-reactive (lysines). PEG spacer arm increases solubility of biotinylated molecule. Maleimide-PEG2-Biotin (Product # 21901, 21902), plus Immobilized TCEP (Product # 77712) Sulfhydryl-reactive. Choose if cysteines are not required for binding reactions (often a terminal cysteine is added during peptide synthesis for this purpose). Must ensure that peptide is reduced. May be difficult to remove unreacted reagent. Amine-PEG2-Biotin (Product # 21346), plus EDC (Product # 22980) and Sulfo-NHS (Product # 24510) Carbohydrate and amine-reactive. Use only if some peptide polymerization is acceptable along with biotinylation. Polymerization may be desirable if it creates large enough molecules to recover conjugate by dialysis. Sulfo-NHS-LC-Biotin (Product # 21335, Kit Product # 21435, 21935) Amine-reactive (lysines). Most commonly-used and most general choice. NHS-PEG4-Biotin (Product # 21329, 21330) Amine-reactive (lysines). PEG spacer arm increases solubility of biotinylated molecule. Sulfo-NHS-SS-Biotin (Product # 21331) Amine-reactive (lysines). Cleavable disulfide spacer allows recovery of biotinylated protein from immobilized NeutrAvidin Protein. Cell Surface Protein Isolation Kit (Product # 89888) Amine-reactive (lysines). Cleavable disulfide spacer allows recovery of biotinylated protein from immobilized NeutrAvidin™ Protein. Psoralen-PEG3-Biotin (Product # 29986) Provides random labeling of nucleic acid backbone. May cause steric hindrance for hybridization. methods. Amine-PEG2-Biotin (Product # 21346), plus EDC (Product # 22980) and Sulfo-NHS (Product # 24510) Requires intact 5'-phosphate; produces end-labeled oligo. For more information, or to download product instructions, visit www.thermo.com/pierce Selection Guide 2 Reactive Group Reacts With Linkage Formed NHS-Ester/Sulfo-NHS Ester Primary Amine (lysine residue) Amide Bond O O Biotin C O N Maleimide Sulfhydryl (cysteine residue – not disulfide bonded) O N O Thioether Bond O Thioether Bond O O C CH2 I C Biotin Pyridyl Disulfide Biotin S S S CH2 Protein Disulfide Bond Biotin S S Protein N Amine Biotin Protein S O SH Protein H N Biotin Iodoacetyl Biotin Protein NH2 Protein O Biotin O NH C Biotin Amide Bond Carboxyl (glutamate or aspartate residues) O NH2 Protein C OH O NH C Biotin Protein (Reaction requires EDC cross-linker) Hydrazide Oxidized Carbohydrate Biotin Hydrazone Bond O O O C NH NH2 Protein C H Biotin H C N N C Protein H Azido (Photoactivatable) NO2 Biotin N H N3 DNA/RNA, Protein, Carbohydrates N Biotin NH Protein Ring expansion followed by coupling with primary amine or insertion into double bonds To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor. 3 Amine-Reactive Biotinylation Reagents Biotinylation Reagents Label almost anything with biotin using one of our many high-quality biotinylation reagents Whether you are an expert or are trying biotinylation for the first time, Thermo Scientific EZ-Link Reagents and Kits make biotinylation easy. Our customers depend on our consistent lotto-lot quality for reproducible results. We offer complete EZ-Link® Biotinylation Kits with buffers, fast and efficient spin desalting columns and HABA detection reagent to determine the number of biotin residues attached to your protein. Amine-Reactive Biotinylation Reagents The most common target for modifying protein molecules is the amine group, which is present on the vast majority of proteins due to the abundance of lysine side chain e-amines and N-terminal a-amines. Based on water solubility, amine-reactive biotinylation reagents can be divided into two groups. NHS-esters of biotin are essentially water-insoluble. For reactions in aqueous solution, they must first be dissolved in an organic solvent, then diluted into the aqueous reaction mixture. The most commonly used organic solvents for this purpose are DMSO and DMF, which are compatible with most proteins at 20% final concentration. The solvent acts as a carrier for the biotinylation reagent, forming a microemulsion in the aqueous phase and allowing the biotinylation reaction to proceed. The water-insoluble NHS-esters of biotin are membrane-permeable because they do not possess a charged group. They may be used for biotinylating internal as well as external components of a cell. Sulfo-NHS-esters of biotin are soluble up to approximately 10 mM in water. Sulfo-NHS-esters should be dissolved in water just before use because they are prone to hydrolysis. The water solubility of sulfo-NHS-esters results from the presence of the sulfonate (–SO3) group on the N-hydroxysuccinimide ring and eliminates the need to dissolve the reagent in an organic solvent. These compounds are used for applications that cannot tolerate organic solvents. Sulfo-NHS-esters of biotin are also recommended for use as cell surface biotinylation reagents. Because of the charged sulfonate group, these compounds do not penetrate the plasma membrane; thus biotinylation is restricted to the cell surface. O O HN O O N There is considerable flexibility in the actual conditions used for conjugating NHS-esters (or sulfo-NHS-esters) to primary amines. Incubation temperatures range from 4-37°C, reaction mixture pH values range from 7-9, and incubation times range from a few minutes to overnight. Buffers containing amines (such as Tris or glycine) must be avoided because they compete with the reaction. In preparing an NHS-ester biotin conjugate, a particular set of conditions will result in a conjugate with optimum properties for a specific application. The preparation of an optimum conjugate is largely dependent on the degree of incorporation of the label. Because of the variability among proteins, especially the number of amines available for conjugation, conjugation conditions that are optimal for one protein may not be optimal for another protein. There are several additional features to consider in an amine-reactive biotinylation reagent. Because biotin binds in a pocket located 9 Å below the surface of the avidin molecule, the spacer arm connected to the biotin is critical. Long spacer arms reduce steric hindrance and result in enhanced interaction of avidin and biotin. It is sometimes necessary to remove biotin from a molecule once a procedure has been completed, and there are reagents with a cleavable spacer arm that allow this procedure. A macromolecule is first reacted with the cleavable biotinylation reagent, then it is used in a detection or purification system. Finally the biotin moiety can be cleaved away, releasing the molecule into solution. Biotin labeling generally reduces the solubility of a molecule and may result in precipitation. Unlike sulfo-NHS-esters that lose their solubility-enhancing sulfonate group during the reaction process, reagents containing a PEG spacer arm retain their high solubility when bound to a protein and are an ideal choice when precipitation must be overcome. O O Na S The reaction chemistries of NHS- and sulfo-NHS-esters are essentially identical: an amide bond is formed and NHS or sulfoNHS are leaving groups in the reaction. Because the target for the ester is the deprotonated form of the primary amine, the reaction becomes significant at neutral pH values and above when the amine is able to react with the ester by nucleophilic attack. Hydrolysis of the NHS-ester is a major competing reaction, and the rate of hydrolysis increases with increasing pH. NHS- and sulfo-NHS-esters have a half-life of hydrolysis of 2-4 hours at pH 7. This half-life decreases to just a few minutes at pH 9. H N O O NH Na S O S O O Sulfo-NHS-LC-Biotin O O N OH O Sulfo-NHS Leaving Group O HN O Protein Protein NH2 Molecule with Primary Amines Reaction of Sulfo-NHS-LC-Biotin with a primary amine. 4 For more information, or to download product instructions, visit www.thermo.com/pierce N H H N NH S O Biotinylated Molecule Thermo Scientific EZ-Link Amine-Reactive Biotinylation Kits Kit Sulfo-NHS Biotinylation Kits Highlights References • Complete kit with an optimized 1, 2 protocol for labeling a protein and determining how much biotin has been attached • Fast and eficient labeling procedure Sulfo-NHS-LC Biotinylation Kits • Complete kit with an optimized 1, 2 protocol for labeling a protein and determining how much biotin has been attached Ordering Information Product # Description Pkg. Size 21425 EZ-Link Sulfo-NHS Biotinylation Kit Kit 21925 EZ-Link Micro Sulfo-NHS Biotinylation Kit Kit 21435 EZ-Link Sulfo-NHS-LC Biotinylation Kits Kit 21935 EZ-Link Micro Sulfo-NHS-LC Biotinylation Kit Kit Includes: No-Weigh Sulfo-NHS-LC Biotin Zeba Desalting Columns BupH Phosphate Buffered Saline 8 x 1 mg 2 ml 1 pack (500 ml) 21455 EZ-Link NHS-PEG4 Biotinylation Kit Kit 21955 EZ-Link Micro NHS-PEG4 Biotinylation Kit Kit Includes: No-Weigh NHS-PEG4-Biotin Zeba Desalting Columns BupH Phosphate Buffered Saline 8 x 1 mg 2 ml 1 pack (500 ml) 21445 EZ-Link Sulfo-NHS-SS Biotinylation Kit Kit 21945 EZ-Link Micro Sulfo-NHS-SS Biotinylation Kit Kit • Fast and eficient labeling procedure NHS-PEG4 Biotinylation Kits Sulfo-NHS-SS Biotinylation Kits • PEG4-Biotin transfers water-solubility to biotinylated molecules • Cleavable biotinylation reagent allows removal of biotin label 3-6 7-9 Includes: Sulfo-NHS-Biotin Reaction Buffer Zeba™ Spin Columns HABA Dye (10 mM) Avidin, affinity-purified Includes: No-Weigh™ Sulfo-NHS-Biotin Zeba Desalting Columns BupH™ Phosphate Buffered Saline Includes: Sulfo-NHS-LC-Biotin Reaction Buffer Zeba Spin Columns HABA Dye (10 mM) Avidin, affinity-purified Includes: NHS-PEG4-Biotin BupH PBS Zeba Spin Columns HABA Dye (10 mM) Avidin, a ffinity-purified Includes: Sulfo-NHS-SS-Biotin Non-reagent contents same as Product # 21425 Includes: Sulfo-NHS-SS-Biotin Non-reagent contents same as Product # 21925 25 mg 1 pack (500 ml) 10 1 ml 10 mg 8 x 1 mg 2 ml 1 pack (500 ml) 25 mg 1 pack (500 ml) 10 1 ml 10 mg 8 x 2 mg 1 pack (500 ml) 10 1 ml 10 mg 25 mg 8 x 1 mg References 1. Zhang, L., et al. (1999). J. Biol. Chem. 274, 8966-8972. 2. Zuk, P.A. and Elferink, L.A. (2000). J. Biol. Chem. 275, 26754-26764. 3. Ali, M.K. and Bergson, C. (2003). J. Biol. Chem. 278, 51654-51663. 4. Du, J., et al. (2003). J. Cell Biol. 163(2), 385-395. 5. Lee-Kwon, W., et al. (2003). J. Biol. Chem. 278, 16494-16501. 6. Lin, Z., et al. (2003). J. Biol. Chem. 278(22), 20162-20170. 7. Daniels, G.M. and Amara, S.G. (1998). Methods. Enzymol. 296, 307-318. 8. Huh, K-H. and Wenthold, R.J. (1999). J. Biol. Chem. 274, 151-157. 9. Trotti, D., et al. (2001). J. Biol. Chem. 276, 576-582. To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor. 5 Amine-Reactive Biotinylation Reagents Thermo Scientific EZ-Link Amine-Reactive Biotinylation Reagents Reagent Structure Highlights Sulfo-NHS-Biotin • Reacts with primary amines at ph 7-9 in non-amine-containing buffers O NH HN • Water-soluble for ease of use O Na+ O- N O S O O S O O Sulfo-NHS-Biotin MW 443.43 Spacer Arm 13.5 Å Sulfo-NHS-LC-Biotin O HN O Na+ OO S N O • Water-soluble for ease of use O H N O • Ideal labeling reagent for monoclonal and polyclonal antibodies NH S O O Sulfo-NHS-LC-Biotin MW 556.59 Spacer Arm 22.4 Å Sulfo-NHS-LC-LC-Biotin O NH HN Na+ O- O O N O S O O O H N N H O • Contains an extra-long spacer arm (30.5 Å) to reduce steric hindrance S O Sulfo-NHS-LC-LC-Biotin MW 669.75 Spacer Arm 30.5 Å Sulfo-NHS-SS-Biotin HN Na+ OO S • Disulide linkage can be cleaved by reducing agents O O N O O O S S H N NH • Ideal reagent for reversibly biotinylating cell surface proteins S O O Sulfo-NHS-SS-Biotin MW 606.69 Spacer Arm 24.3 Å NHS-PEG4-Biotin O HN O N O O O O O O O H N NH • PEG spacer arm increases solubility of conjugates • No-Weigh Packaging ensures fresh reagent and eliminates tedious weighing of reagent S O NHS-PEG4-Biotin MW 588.67 Spacer Arm 29.0 Å 6 For more information, or to download product instructions, visit www.thermo.com/pierce References Ordering Information Product # Description Pkg. Size 21217 EZ-Link Sulfo-NHS-Biotin 50 mg 21326 No-Weigh Sulfo-NHS-Biotin 8 x 1 mg • Arosa, F.A., et al. (1999). J. Biol. Chem. 274, 16917-16922. • Baqui, M., et al. (2003). J. Biol. Chem. 278, 1206-1211. • Huh, K-H. and Wenthold, R.J. (1999). J. Biol. Chem. 274, 151-157. • Lesa, G.M., et al. (2000). J. Biol. Chem. 275, 2831-2836. • Liaw, P.C.Y., et al. (2001). J. Biol Chem. 276, 8364-8370. • Liu, L.A. and Engvall, E. (1999). J. Biol. Chem. 274, 38171-38176. 21335 EZ-Link Sulfo-NHS-LC-Biotin 100 mg 21327 No-Weigh Sulfo-NHS-LC-Biotin 8 x 1 mg • Muroi, M., et al. (2002). J. Biol. Chem. 277, 42372-42379. • Schwarzman, A.L., et al. (1999). Proc. Natl. Acad. Sci. U.S.A. 96, 7932-7937. 21338 EZ-Link Sulfo-NHS-LC-LC-Biotin 50 mg • Daniels, G.M. and Amara, S.G. (1998). Methods. Enzymol. 296, 307-318. • Huh, K-H. and Wenthold, R.J. (1999). J. Biol. Chem. 274, 151-157. • Trotti, D., et al. (2001). J. Biol. Chem. 276, 576-582. 21331 EZ-Link Sulfo-NHS-SS-Biotin 100 mg 21328 No-Weigh Sulfo-NHS-SS-Biotin 8 x 1 mg 21329 No-Weigh NHS-PEG4-Biotin 8 x 2 mg 21330 EZ-Link NHS-PEG4-Biotin 25 mg 21362 EZ-Link NHS-PEG4-Biotin 50 mg 21363 EZ-Link NHS-PEG4-Biotin 1g • Arosa, F.A., et al. (1999). J. Biol. Chem. 274, 16917-16922. • Claypool, S.M., et al. (2002). J. Biol. Chem. 277, 28038-28050. • Ellerbroek, S.M., et al. (2001). J. Biol. Chem. 276, 24833-24842. • Leighton, B.H., et al. (2002). J. Biol. Chem. 277, 29847-29855. • Neely, K.E., et al. (2002). Mol. Cell. Biol. 22, 1615-1625. • Dodeller, F., et al. (2008) J. Biol. Chem. 283, 21487-21494. • Newton, J.R., et al. (2007) J. Nucl. Med. 48, 429-436. • Behrens, M. et al. (2006) J. Biol. Chem. 281, 20650-20659. To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor. 7 Amine-Reactive Biotinylation Reagents Thermo Scientific EZ-Link Amine-Reactive Biotinylation Reagents Reagent Structure Highlights NHS-PEG12-Biotin • PEG spacer arm increases solubility of conjugates O HN O N NH O [ O O O ] H N 12 • Extra long spacer arm reduces steric hindrance S O NHS-PEG12-Biotin MW 941.09 Spacer Arm 56 Å NHS-SS-PEG4-Biotin O HN O N S O O H N S O O O H N O S • PEG spacer arm increases solubility of conjugates • Disulide linkage can be cleaved by reducing agents O O O NH NHS-SS-PEG4-Biotin M.W. 751.94 Spacer Arm 37.9 Å NHS-Biotin • Must be dissolved in DMSO or DMF before adding to aqueous solution O HN NH • Able to penetrate cell membranes O N O O S O NHS-Biotin MW 341.38 Spacer Arm 13.5 Å NHS-LC-Biotin O O N O O HN O H N NH • Must be dissolved in DMSO or DMF before adding to aqueous solution • Able to penetrate cell membranes S O NHS-LC-Biotin MW 454.54 Spacer Arm 22.4 Å 8 For more information, or to download product instructions, visit www.thermo.com/pierce References Ordering Information Product # Description Pkg. Size 21312 EZ-Link NHS-PEG12-Biotin 25 mg 21313 EZ-Link NHS-PEG12-Biotin 500 mg 21442 NHS-SS-PEG4-Biotin 50 mg • Fouassier, L., et al. (2000). J. Biol. Chem. 275, 25039-25045. • Nunomura, W., et al. (2000). J. Biol. Chem. 275, 6360-6367. 20217 EZ-Link NHS-Biotin 100 mg • Chiu, N.H., et al. (1999). Clin. Chem. 45, 1954-1959. • Schumacher, T.N., et al. (1996). Science 271, 1854-1857. • Tang, A., et al. (1993). Nature 361, 82-85. 21336 EZ-Link NHS-LC-Biotin 50 mg To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor. 9 Amine-Reactive Biotinylation Reagents Thermo Scientific EZ-Link Amine-Reactive Biotinylation Reagents Reagent Structure Highlights NHS-LC-LC-Biotin HN O O O N O • Contains an extra-long spacer arm (30.5 Å) to reduce steric hindrance O H N N H O NH S O NHS-LC-LC-Biotin MW 567.70 Spacer Arm 30.5 Å NHS-SS-Biotin O NH HN O N S O H N S O O • Label almost any protein with biotin to facilitate immobilization, purification or detection • Disulide bond in the spacer arm allows the biotin label to be removed S • Amine-reactive NHS ester reacts rapidly with any primary aminecontaining molecule to attach the biotin via a stable amide bond O NHS-SS-Biotin • Long spacer arm on the biotin reduces steric hindrance with binding to avidin or other biotin-binding proteins MW 504.65 Spacer Arm 24.3 Å NHS-Iminobiotin O • Reversible binding to avidin prevents protein denaturation during purification F N F F NH HN • Binds to avidin at pH >9.5 and dissociates at pH 4 O N O O S O NHS-Iminobiotin Trifluoroacetamide MW 436.41 Spacer Arm 13.5 Å PFP-Biotin • More reactive than NHS esters O HN • Will react with primary and secondary amines at pH 7-9 NH • Easier-to-handle and store than NHS esters F O F F F • Great for DNA labeling S • PEG spacer arm increases solubility of conjugates O F Biotin-PFP Ester MW 410.36 Spacer Arm 9.6 Å TFP-PEG3-Biotin F F F HN O H N O F • Long-chain, water-soluble, polyethylene oxide (PEG) spacer arm O O O O O NH • Tetraluorophenyl (TFP) ester reacts with primary and secondary amines at pH 7-9 • Glycine and Tris buffers interfere with reaction H N S • PEG spacer arm transfers hydrophilicity to inal conjugate O TFP-PEG3-Biotin MW 694.74 Spacer Arm 32.6 Å 10 For more information, or to download product instructions, visit www.thermo.com/pierce References Ordering Information Product # Description Pkg. Size 21343 EZ-Link NHS-LC-LC-Biotin 50 mg 21441 EZ-Link NHS-SS-Biotin 50 mg • Orr, G.A., et al. (1981). J. Biol. Chem. 256, 761-766. • Zeheb, R., et al. (1983). Anal. Biochem. 129, 156-161. 21117 EZ-Link NHS-Iminobiotin 100 mg • Muroi, M., et al. (2002). J. Biol. Chem. 277, 42372-42379. • Michaelis, K., et al. (2006) J. Pharmacol. Exp. Ther. 317, 1246-1253. 21218 EZ-Link PFP-Biotin 50 mg 21219 EZ-Link TFP-PEG3-Biotin 50 mg • Bradley, C., et al. (2007) Carcinogen. 28, 2184-2192. • Sehr, P., et al. (2007) J. Biomo.l Screen. 12, 560-567. • Negishi, A., et al. (2004) Glycobiolog. 14, 969-977. To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor. 11 Sulfhydryl-Reactive Biotinylation Reagents Sulfhydryl-Reactive Biotinylation Reagents The second most common target for modification in biological molecules is free sulfhydryl groups, which are found in the form of exposed cysteine in a protein or peptide. Sulfhydryl group biotinylation may provide an advantage in some applications; for example, targeting a sulfhydryl group can be used as a method for preserving the biological activity of an enzyme when amines are found at the active site. Modification of these amines may render the enzyme inactive. This complication can be avoided by using derivatives of biotin that react with sulfhydryls. Because biotinylation with these reagents must be performed in buffers free of extraneous sulfhydryls, substances such as 2-mercaptoethanol, dithiothreitol and mercaptoethylamine must be removed before biotinylation. can be generated from disulfides by incubation with a reducing agent or from lysine residues by incubating with modification reagents such as 2-Iminothiolane (Traut’s Reagent) or SATA. When working with free sulfhydryls, EDTA is generally included in the buffer system for its antioxidative effect. EDTA chelates trace amounts of metals in solution that promote disulfide bond formation. Using nitrogen-purged buffers is an additional precaution to prevent oxidation of the free sulfhydryls. Three separate reaction chemistries are employed to target sulfhydryl groups for biotinylation. The most specific method uses reactive maleimide groups, which are 1,000 times more reactive toward free sulfhydryls than toward amines at pH 7. Biotin-BMCC must be dissolved in an organic solvent, then diluted into an aqueous reaction mixture. Maleimide-PEG2-Biotin, which is watersoluble by virtue of its polyethyleneoxide (PEG) spacer arm, may be dissolved directly in aqueous solution. The reaction of maleimide with free thiols is carried out at pH 6.5-7.5 because cross-reactivity Proteins, peptides or other molecules to be biotinylated by sulfhydryl-reactive reagents must have a free sulfhydryl group (–SH) available; disulides will not react with sulfhydryl-speciic biotinylation reagents. If free sulfhydryls are not available, they Thermo Scientific EZ-Link Sulfhydryl-Reactive Biotinylation Reagents Reagent Structure Highlights Maleimide-PEG2-Biotin O • Water-soluble NH HN O O N O N H H N O S • Reacts with sulfhydryl (–SH) groups at acidic to neutral pH • Avoids potential modiication of tyrosine residues that can be associated with Iodoacetyl-Biotin O O Maleimide-PEG2-Biotin MW 525.62 Spacer Arm 29.1 Å Maleimide-PEG11-Biotin O HN O H N N O • Water-soluble NH O [ O ] H N 11 • Reacts with sulfhydryl (-SH) groups at acidic to neutral pH • Extra long spacer arm reduces steric hindrance S O Maleimide-PEG11-Biotin MW 922.09 Spacer Arm 59.1 Å Biotin-BMCC • Reacts with sulfhydryl (–SH) groups at pH 6.5-7.5 O NH HN O O H N N H N S • Avoids potential modiication of tyrosine residues that can be associated with Iodoacetyl-Biotin • Must be dissolved in DMSO or DMF before use O Biotin-BMCC O MW 533.68 Spacer Arm 32.6 Å Iodoacetyl-PEG2-Biotin HN O I • Water-soluble O N H O O H N NH • Reacts with sulfhydryl (–SH) groups at basic to neutral pH • Coupling reactions occur in the dark S O Iodoacetyl-PEG2-Biotin MW 542.43 Spacer Arm 24.7 Å 12 For more information, or to download product instructions, visit www.thermo.com/pierce toward primary amines can occur at higher pH values. Hydrolysis of the maleimide group also increases at higher pH values. Iodoacetyl groups are also used to target free sulfhydryls. Iodoacetyl-LC-Biotin is not water-soluble and must be dissolved in a solvent before use in an aqueous reaction mixture. IodoacetylPEG2-Biotin, which is water-soluble by virtue of its PEG spacer arm, may be dissolved directly in aqueous solution. The iodoacetyl group reacts mainly with thiol groups at pH 7.5-8.5, resulting in a stable thioether bond. Unless precautions are taken, iodoacetyl groups may not be specific for sulfhydryls. The reaction can be directed toward sulfhydryl groups by limiting the molar ratio of IodoacetylBiotin to protein, such that the concentration of biotin is present at a small excess over the sulfhydryl content. Also, the reaction pH should be maintained in the range of 7.5-8.5. Below pH 9, crossreactivity with amine, thioether and imidazole groups is minimized. Therefore, maintaining a lower pH ensures the modification of sulfhydryl groups and not amino groups. If there are no cysteines References available, the reaction can be directed at imidazoles by adjusting the pH to 6.9-7.0. However, the incubation time must be increased to a week. Histidyl side chains and amino groups react in the deprotonated form and may take part in reactions above pH 5 and pH 7, respectively, although this reaction is much slower than that for sulfhydryls. A pyridyldithiol group may also be used to attach biotin to a free sulfhydryl by disulfide exchange, resulting in the formation of a mixed disulfide bond. The reaction of Biotin-HPDP is generally carried out under physiologic conditions although the process occurs within a wide range of pH conditions and with a variety of buffer components. Pyridyldithiol reactions result in the release of pyridine-2-thione, which cannot react with free sulfhydryls. The release of this compound can be measured and the reaction progress monitored by an increasing absorbance at 343 nm. Another important feature of this method is that the biotin is released by treatment with reducing agents. Ordering Information Product # Description Pkg. Size 21901 EZ-Link Maleimide-PEG2-Biotin 50 mg 21902 No-Weigh Maleimide-PEG2-Biotin Microtubes 8 x 2 mg 21911 Maleimide-PEG11-Biotin 25 mg • Roberts, P.J., et al. (2008) J. Biol. Chem. 283, 25150-25163. • Shi, M., et al. (2007) J. Biol. Chem. 282, 30198-30206. • Chenette, E. J., et al. (2006) Mol. Biol. Cell. 17, 3108-3121. 21900 EZ-Link Biotin-BMCC 50 mg • Kim, K., et al. (2001). J. Biol. Chem. 276, 40591-40598. • Muroi, M., et al. (2002). J. Biol. Chem. 277, 42372-42379. 21334 EZ-Link Iodoacetyl-PEG2-Biotin 50 mg • Oda, Y., et al. (2001). Nat. Biotechnol. 19, 379-382. • Inglis, K.J., et al. (2008) J. Biol. Chem. doi:10.1074/jbc.C800206200 To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor. 13 Sulfhydryl-Reactive Biotinylation Reagents Thermo Scientific EZ-Link Sulfhydryl-Reactive Biotinylation Reagents Reagent Structure Highlights Iodoacetyl-LC-Biotin • Reacts with sulfhydryl (–SH) groups at basic to neutral pH O HN O I H N N H • Coupling reactions occur in the dark NH • Must be dissolved in DMSO or DMF before use S O Iodoacetyl-LC-Biotin MW 510.43 Spacer Arm 27.1 Å Biotin-HPDP O HN O N S S H N N H S O Biotin-HPDP NH • Pyridyldithiol reacts with sulfhydryl (–SH) groups to form a stable disulfide bond between pH 6-9 • Pyridine-2-thione leaving group can be used to measure degree of biotinylation at 343 nm • Disulide bond is cleavable using 50 mM DTT or 100 mM 2-mercaptoethanol • Must be dissolved in DMSO or DMF before use MW 539.78 Spacer Arm 29.2 Å 14 For more information, or to download product instructions, visit www.thermo.com/pierce References Ordering Information Product # Description Pkg. Size • Sutoh, K., et al. (1984). J. Mol. Biol. 178, 323-339. • Yamamoto, K., et al. (1984). FEBS Lett. 176, 75-78. 21333 EZ-Link Iodoacetyl-LC-Biotin 50 mg • Ishmael, F.T., et al. (2001). J. Biol. Chem. 276, 25236-25242. • Slatin, S.L., et al. (2002). Proc. Natl. Acad. Sci. U.S.A. 99, 1286-1291. 21341 EZ-Link Biotin-HPDP 50 mg To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor. 15 Carboxyl-Reactive Biotinylation Reagents Carboxyl-Reactive Biotinylation Reagents Carboxyl groups, in the form of carboxy termini, aspartate residues or glutamate residues, can also be targeted for biotin labeling using amine-derivatized biotin molecules. This reaction is mediated by a class of crosslinkers known as carbodiimides and results in the formation of an amide bond. The reaction with EDC, the most common carbodiimide crosslinker, is generally performed in an MES buffer at pH 4.5-5 and requires just minutes to complete. Buffers containing primary amines (Tris, glycine, etc.) or carboxyls (acetate, citrate, etc.) must be avoided because they will quench the reaction. Phosphate buffers are also not recommended because they reduce the conjugation efficiency, although this effect can be overcome by adding more EDC. Thermo Scientific EZ-Link Carboxyl-Reactive Biotinylation Reagents Reagent Structure Highlights Pentylamine-Biotin • Use the EDC crosslinker to couple this analog to carboxyl (–COOH) groups O HN H N H2N NH • Dissolves in aqueous solutions • Coupling with EDC occurs at pH 4-6 S O Pentylamine-Biotin MW 328.47 Spacer Arm 18.9 Å Amine-PEG2-Biotin • Use the EDC crosslinker to couple this analog to carboxyl (–COOH) groups O NH HN O H2N H N O • Water-soluble S O Amine-PEG2-Biotin MW 374.50 Spacer Arm 20.4 Å Amine-PEG3-Biotin O HN H2N O O O H N NH S • Use the EDC crosslinker to couple this analog to carboxyl (–COOH) groups • Water-soluble • Longer chain length for reduction of steric hindrance O Amine-PEG3-Biotin MW 418.55 Spacer Arm 22.9 Å 16 For more information, or to download product instructions, visit www.thermo.com/pierce Amine-PEG2-Biotin, Amine-PEG3-Biotin and Pentylamine-Biotin are amine-derivatized biotin molecules that can be reacted with carboxyl groups. In addition, any of the hydrazide-derivatized biotin molecules can be reacted with carboxyls under identical conditions. The reaction is most often mediated by EDC, a water-soluble carbodiimide that activates carboxyl groups to bind to the –NH2 group on the biotinylation reagent. Using this strategy may result in some polymerization of the peptide or protein if the molecule has both carboxyls and primary amines on its surface. The extent of polymerization can be minimized by decreasing the amount of EDC and/or increasing the amount of the biotin reagent used in the reaction. References Ordering Information Product # Description Pkg. Size • Cernuda-Morollon, E., et al. (2001). J. Biol. Chem. 276, 35530-35536. • Liu, Y., et al. (1999). Proc. Natl. Acad. Sci. U.S.A. 96, 14694-14699. 21345 EZ-Link Pentylamine-Biotin 50 mg • Fezza, F., et al. (2008) J. Lipid Res. 49, 1216-1223. • Cui, B., et al. (2007) Proc. Nat. Acad. Sci. USA. 104, 13666-13671. • Pihlajamaa, T., et al. (2004) J. Biol. Chem. 279, 24265-24273. 21346 EZ-Link Amine-PEG2-Biotin 50 mg • Maguire, B. A., et al. (2008) RNA. 14, 188-195. 21347 EZ-Link Amine-PEG3-Biotin 50 mg To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor. 17 Carbohydrate/Aldehyde-Reactive Biotinylation Reagents Carbohydrate/Aldehyde-Reactive Biotinylation Reagents Another common target for protein modification is the carbohydrate portion of glycoproteins, which can be reacted with hydrazidederivatives of biotin. Oxidative treatment of glycoproteins using 10 mM periodate is used to generate reactive aldehydes from the cis-diols of a variety of carbohydrate moieties. OH OH R C C H H O NaIO4 R' R C An aldehyde can be reacted specifically with a hydrazide group at pH 4-6, forming a stable hydrazone linkage. Sialic acid residues on glycoproteins can be specifically oxidized with sodium periodate (NaIO4) under mild conditions. At 1 mM periodate and a temperature of 0°C, oxidation is restricted primarily to sialic acid residues. Sialic acid residues also can be biotinylated with hydrazide derivatives by pretreatment with neuraminidase to generate galactose groups. The galactose and N-acetylgalactosamine residues on whole cells can be selectively biotinylated with Biotin-Hydrazides by further treatment with galactose oxidase. This enzyme will convert the primary hydroxyl groups on these sugars to their corresponding aldehydes. O H + H C R' Oxidation of a cis-diol to an aldehyde Thermo Scientific EZ-Link Carbohydrate/Aldehyde-Reactive Biotinylation Reagents Reagent Structure Highlights Biocytin-Hydrazide • Can be used to label DNA and RNA through cytosine residues O NH HN O H2N N H H N NH2 • More water-soluble than Biotin-LC-Hydrazide S O Biocytin-Hydrazide MW 386.51 Spacer Arm 19.7 Å Biotin-Hydrazide • Carbohydrate reactive O H 2N H N • Must be dissolved in DMSO before adding to aqueous buffer NH HN S O Biotin-Hydrazide MW 258.34 Spacer Arm 15.7 Å Biotin-LC-Hydrazide O HN O H2N H N N H • Use the EDC crosslinker to couple this analog to carboxyl (–COOH) groups NH • Water-soluble • Longer chain length for reduction of steric hindrance S O Biotin-LC-Hydrazide MW 371.50 Spacer Arm 24.7 Å Biotin-PEG4-Hydrazide HN H2N H N • Water-soluble analog of Biotin-LC-Hydrazide O O O O O O H N NH • PEG-based spacer arm increases solubility of labeled molecules S O Biotin-PEG4-Hydrazide MW 505.63 Spacer Arm 31.3 Å 18 For more information, or to download product instructions, visit www.thermo.com/pierce Mild oxidation of an immunoglobulin with sodium periodate produces reactive aldehydes from the carbohydrate moieties on the Fc portion of the antibody, which then can be alkylated by a hydrazide. This approach is advantageous for use with antibodies because they become biotinylated in a manner that maintains immunological reactivity. This is an ideal method for biotinylating polyclonal antibodies because they are heavily glycosylated. Monoclonal antibodies may be deficient in glycosylation and success with this method will depend on the extent of glycosylation for a particular antibody. References Temperature, pH of oxidation and the periodate concentration all affect the reaction with hydrazide derivatives of biotin. Also, because glycosylation varies with each protein, optimum conditions must be determined for each glycoprotein. Each glycoprotein preparation has an optimum pH for oxidation and for the hydrazide-mediated biotinylation. Tris, or other primary amine-containing buffers, are not recommended for use in either the oxidation or biotinylation steps because these buffers react with aldehydes, quenching their reaction with hydrazides. Ordering Information Product # Description Pkg. Size • Bayer, E.A. et al. (1988). Anal. Biochem. 170, 271-281. • Reisfeld, A., et al. (1987). Biochem. Biophys. Res. Commun. 142, 519-526. • Roffman, E., et al. (1986). Biochem. Biophys. Res. Commun. 136, 80-85. 28020 EZ-Link Biocytin-Hydrazide 25 mg • Edwards, S.W., et al. (1999). J. Biol. Chem. 274, 16331-16336. • Reisfeld, A., et al. (1987). Biochem. Biophys. Res. Commun. 142, 519-526. 21339 EZ-Link Biotin-Hydrazide 100 mg • Araga, S., et al. (1999). J. Immunol. 163, 476-482 21340 EZ-Link Biotin-LC-Hydrazide 50 mg 21360 EZ-Link Biotin-PEG4-Hydrazide 50 mg • • • • Kahne, T. and Ansorge, S. (1994). J. Immunol. Methods 168, 209-218. Luk, J.M., et al. (1995). Anal. Biochem. 232, 217-224 Scott, M.G., et al. (2000). J. Immunol. 164, 549-553. Yu, Q. and Toole, B.P. (1995). Biotechniques 19, 122-129. To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor. 19 Photo-Reactive Biotinylation Reagents Photo-Reactive Biotinylation Reagents Proteins, peptides and other molecules that do not contain any of the reactive functional groups mentioned previously may be labeled using a photo-reactive biotinylation reagent. These reagents contain an aryl azide group that is chemically inert until it is exposed to ultraviolet light, causing the formation of a short-lived, reactive aryl nitrene. The half-life of this aryl nitrene intermediate is on the order of 10-4 seconds. The aryl nitrene reacts rapidly and nonselectively with electron dense sites by addition into double bonds or insertion into active hydrogen bonds. If the aryl-nitrene fails to react, it undergoes ring expansion and becomes reactive toward nucleophiles such as primary amines and sulfhydryls. Photoactivation and insertion into another molecule can be performed in a wide variety of buffer conditions. However, acidic conditions and reducing agents should be avoided because they may inactive the aryl azide group. Thermo Scientific EZ-Link Photo-Reactive Biotinylation Reagents Reagent Structure Highlights Photoactivatable Biotin -O -N N+ • Covalent attachment occurs in the presence of UV light (350-370 nm) O N+ NH HN O CH3 H N H N N • Attachment is nonspeciic • Must be dissolved in DMSO or DMF before use S • Reaction buffer should be above pH 6 O N Photoactivatable Biotin MW 533.65 Spacer Arm 30.0 Å Psoralen-PEG3-Biotin • Water-soluble O O NH HN O H N O O O O H N O • Psoralen moiety reacts with the 5,6 double bond in thymineand other pyrimidine-containing bases S • Covalent attachment occurs in the presence of UV light (>350 nm) for 10-30 minutes O O • Much higher coupling yield than achieved with photoactivatable biotin Psoralen-PEG3-Biotin • Great for biotinylating DNA/RNA probes MW 688.79 Spacer Arm 36.9 Å • Ultrahigh sensitivity relative to radiolabeled probes (<100 femtograms) • Doesn’t interfere with hybridization Biotin-LC-ASA • Iodinatable and photoactivatable O HN OH -N N+ O H N N H • Useful for biotinylation of nucleic acids NH S O N Biotin-LC-ASA MW 503.62 Spacer Arm 29.9 Å TFPA-PEG3-Biotin F + -N N HN F N H N F F • Label almost any molecule with biotin O O O O O H N NH • TFPA is more eficient than other photo-reactive groups • Long, PEG-based spacer arm reduces steric hindrance and increases conjugate solubility S O TFPA-PEG3-Biotin MW 663.69 Spacer Arm 33.4 Å 20 For more information, or to download product instructions, visit www.thermo.com/pierce Photoactivatable Biotin, Biotin-LC-ASA , Psoralen-PEG3-Biotin and TFPA-PEG3-Biotin each contain a photoactivatable group. When exposed to UV light, they become activated and insert nonspecifically into nearby molecules. These reagents may be used to label proteins and peptides, but they are also useful in labeling DNA, RNA and other molecules that do not contain any readily reactive functional groups. References Psoralen-PEG3-Biotin contains a reactive psoralen group and is designed to efficiently label nucleic acids. The psoralen group intercalates into the helix of DNA, allowing it to label efficiently and selectively. The psoralen can also stack along with the bases of single-stranded DNA or RNA increasing labeling efficiency and selectivity. Upon photoactivation, psoralen forms a crosslink with the 5,6 double bond of pyrimidine bases and its presence does not interfere with hybridization. Ordering Information Product # Description Pkg. Size • Lacey, E. and Grant, W.N., (1987). Anal. Biochem. 163, 151-158. • Smith, J.S., et al. (1999). Proc. Natl. Acad. Sci. U.S.A. 96, 8855-8860. 29987 EZ-Link Photoactivatable Biotin 0.5 mg • Cimono, G.D., et al. (1985). Annu. Rev. Biochem. 54, 1151-1193. 29986 EZ-Link Psoralen-PEG3-Biotin 5 mg 29982 EZ-Link Biotin-LC-ASA 2 mg 21303 EZ-Link TFPA-PEG3-Biotin 25 mg • Wassarman, D.A. (1993). Mol. Biol. Rep. 17, 143-151. • Kotani, N., et al. (2008) Proc. Nat. Acad. Sci. USA. 105, 7405-7409. • Koraha, J., et al. (2005) Clin. Diagn. Lab. Immunol. 12, 1292-1297. To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor. 21 Specialty Biotinylation Reagents EZ-Link Specialty Biotinylation Reagents Reagent Structure Highlights O Biocytin HN O H N HO • Useful for synthesizing long-chain biotinylation reagents NH S NH2 O Biocytin MW 372.48 Spacer Arm 20.1 Å PEG5-Biotin Dimer HN • Long spacer arm biotin dimer for crosslinking avidin molecules O O HN NH H N S O O O O H N O O NH S • Can be used to increase signal in assays using the avidin-biotin system • Water-soluble O PEG5-Biotin Dimer MW 732.95 Spacer Arm 43.4 Å Label Transfer with Specialty Biotinylation Reagents The label transfer technique is gaining popularity as a valuable strategy for identifying relevant protein interactions. A label transfer reagent consists of a reactive group that can be bound to a protein of interest, a second reactive group that can be bound to another protein that interacts with the protein of interest, a cleavable spacer arm, and a label that can be used to purify or identify the labeled protein. One advantage of the label transfer technique over other in vitro methods for studying protein interactions is its ability to identify molecules with only a weak or transient interaction. These transiently interacting molecules are generally not co-purified using immunoprecipitation or pull-down methods because of the number of washing steps and the time involved. Sulfo-SBED contains an amine-reactive NHS-ester, a photoreactive group, a cleavable disulfide linkage, and a biotin label. Available amine groups on a purified protein are first reacted with the NHS-ester of Sulfo-SBED. The labeled protein is then Thermo Scientific Pierce Specialty Biotinylation Reagents Reagent Structure Highlights Sulfo-SBED Biotin Label Transfer Reagent • Photo-reactive aryl nitrene group couples to protein through C-H bonds when activated at 300-350 nm O HN O Na+ O- O O O S N O O O S S N H H N HN O NH S O • Sulfo-NHS ester reacts with amines to form stable amide bonds at pH 7-9 • Spacer arm contains a biotin group to allow recovery of conjugate on immobilized avidin column • Cleavable disulide bond in the spacer arm allows molecules to be detached from each other Sulfo-SBED MW 879.98 N 22 N+ N For more information, or to download product instructions, visit www.thermo.com/pierce References Ordering Information Product # Description Pkg. Size • Baqui, M., et al. (2003). J. Biol. Chem. 278, 1206-1211. 28022 EZ-Link Biocytin 100 mg • Cimono, G.D., et al. (1985). Annu. Rev. Biochem. 54, 1151-1193. 22020 EZ-Link PEG5-Biotin Dimer 50 mg • Wassarman, D.A. (1993). Mol. Biol. Rep. 17, 143-151. incubated with either a pure preparation of a binding partner or with a complex mixture such as a lysate to search for a binding partner. Complexes form through protein:protein interactions and the sample is exposed to UV light in the 300-366 nm range. The photo-reactive moiety inserts itself into a nearby bond trapping the interacting protein in a covalent complex. By reducing the disulfide linkage, the label can be transferred to the interacting protein. Then the protein can be purified or detected using the biotin label. References Sulfo-SBED Applications include: • Searching for putative binding partners • Interaction mapping • Study of complex assembly mechanisms • Deining docking site and co-factor requirements of interactions • Studying refolding interactions • Detecting low abundance receptors • Evaluating drug-receptor interactions Ordering Information • Alley, S.C., et al. (2000). J. Am. Chem. Soc. 122, 6126-6127. • Daum, J.R., et al. (2000). Curr. Biol. 10, 850-852. • Geselowitz, D.A. and Neumann, R.D., (1995). Bioconjugate Chem. 6, 502-506. • Horney, Mark J., et al. (2001). J. Biol. Chem. 276, 2880-2889. • Ilver, D., et al. (1998). Science 279, 373-377. • Ishmael, F.T., et al. (2002). J. Biol. Chem. 277, 20555-20562. • Jacobson, et al. (1995). Life Sci. 56, 823-830. • Kleene, R., et al. (2000). Biochemistry 39, 9893-9900. • Minami, Y., et al. (2000). J. Biol. Chem. 275, 9055-9061. • Muroi, M., et al. (2002). J. Biol. Chem. 277, 42372-42379. • Neely, K.E., et al. (2002). Mol. Cell. Biol. 22, 1615-1625. • Sharma, K.K., et al. (2000). J. Biol. Chem. 275, 3767-3771. • Trotman, L.C., et al. (2001). Nat. Cell Biol. 3, 1092-1100. Product # Description Pkg. Size 33033 Sulfo-SBED Biotin Label Transfer Reagent † 10 mg 33034 Sulfo-SBED Biotin Label Transfer Reagent 8 x 1 mg 33073 Sulfo-SBED Biotin Label Transfer Kit-Western Blot Application Kit Sufficient reagents for 8 label transfer reactions Includes: Sulfo SBED Phosphate Buffered Saline Label Transfer Buffer (20X) Streptavidin-Horseradish Peroxidase Conjugate Dithiothreitol (DTT) Slide-A-Lyzer® MINI Dialysis Units Plus Float, 10K MWCO 8 x 1 mg 1 pack 200 ml 0.1 mg 8 x 7.7 mg 10 units/pkg. † See patent information. To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor. 23 Specialty Biotinylation Reagents Sulfhydryl-directed, photoactivated biotin label transfer reagents Thermo Scientific Mts-Atf-Biotin Reagents are useful for determining specific components of a protein interaction. A known purified protein can be labeled specifically at native or engineered sulfhydryl sites, and then allowed to bind with its interactors. Exposure to UV-light activates the tetrafluorophenyl azide group, resulting in conjugation of the interacting proteins. After affinity purification, the Thermo Scientific Pierce Specialty Biotinylation Reagents Reagent Structure Highlights Mts-ATF-Biotin Label Transfer Reagent O • Sulfo-NHS ester reacts with primary amines at pH 7-9 • Aryl azide conjugates randomly when activated with UV-light CH3 • Cleavable disulide bond allows biotin label transfer S • Discover protein interactions without radiolabeling S O O HN F HN -N N+ O N O N H F NH HN O H N S O Mts-Atf-Biotin F MW 839.95 F Mts-ATF-Biotin-LC Label Transfer Reagent • Sulfo-NHS ester reacts with primary amines at pH 7-9 • Aryl azide conjugates randomly when activated with UV-light • Cleavable disulide bond allows biotin label transfer CH3 O S O • Discover protein interactions without radiolabeling S O HN HN O HN O N H O O F -N 24 S O Mts-Atf-LC-Biotin NH F F H N NH MW 953.11 F +N N For more information, or to download product instructions, visit www.thermo.com/pierce captured interactors can be released from the original known protein “bait” by reduction of the disulfide bond, leaving a biotin label on the interactor. References Ordering Information • Layer, G. et al. (2007). J. Biol. Chem. 282, 13342-13350. Product # Description Pkg. Size 33083 Mts-ATF-Biotin Label Transfer Reagent 5 mg 33093 Mts-ATF-Biotin-LC Label Transfer Reagent 5 mg To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor. 25 Avidin-Biotin-Based Kits Pierce Biotin Quantitation Kit A convenient, accurate method for determining the degree of biotinylation. Determine the molar ratio of biotin incorporated into an antibody using the HABA-Avidin method. The HABA dye (2-hydroxyazobenzene-4'-carboxylic acid) binds to avidin to produce a yelloworange colored complex that absorbs at 500 nm. Free biotin present in solution with this avidin-HABA complex will displace the HABA dye and cause the absorbance to decrease in proportion to the amount of biotin. The amount of biotin present can be calculated directly from the decreased absorbance at 500 nm. The Thermo Scientific Pierce Biotin Quantitation Kit contains pre-measured doses of the Avidin-HABA mixture and Biotinylated HRP positive control to simplify reagent preparation and minimize the amount of waste generated. The convenient assay can be performed either in a cuvette or in a microplate, and the math is simplified using a calculator on the our web site. Thermo Scientific Pierce Avidin and Biotinylated HRP are also available separately in larger quantities. How does this biotinylation assay work? The HABA4:Avidin complex is at the core of this displacement assay that can estimate the extent of protein biotinylation. HABA dye binds to avidin to form a complex that absorbs strongly at 500 nm with an extinction at that wavelenth of 35,000 M-1cm-1. Highlights: • HABA-avidin complex can be used over a wide range of pH and salt concentrations • Amount of biotin can be calculated directly from the decreased absorbance at 500 nm complexing with the HABA dye Ordering Information Product # Description Pkg. Size Includes: No-Weigh HABA-Avidin Premix Biotinylated HRP Biotin Quantitation Kit 24 assays 24 tubes 5 mg 28010 HABA 10 g 21121 Avidin 10 10 mg 29129 D-Biotin 1g 29139 Biotinylated-HRP 5 mg 28005 (2-[4'-Hydroxyazobenzene]-benzoic acid) References Janolino, V.G., et al. (1996). App. Biochem. Biotech. 56, 1-7. Nikitina, T. and Woodcock, C.L. (2004). J. Cell Biol. 166, 161-165. Savage, M.D., et al. (1992). Avidin-Biotin Chemistry: A Handbook. Rockford, Illinois: Pierce Chemical Company. Zhang, Y. and Pardridge, W. (2005). J. Pharmacol. Exp. Ther. 313, 1075-1081. Hanington, P. et al., (2007). J. Biol. Chem. 282, 31865-31872. The assay is based on the decrease in absorbance of the [(HABA4):Avidin] complex when HABA is displaced from the complex by biotin. [(HABA4):Avidin] λmax = 500 nm ε500nm = 35,500 M-1cm-1 Biotinylated-Protein HABA [Protein-Biotinn:Avidin] 26 For more information, or to download product instructions, visit www.thermo.com/pierce Protein Labeling – Biotinylation Kits Two kit sizes and labeling chemistries: EZ-Link Solid-Phase Biotinylation Kits An easier way to biotinylate IgG antibodies. This innovative antibody-labeling system uses nickel-chelated agarose to temporarily immobilize antibody molecules via their histidine-rich Fc regions. Once held in place on the gel, the antibody can be biotinylated at either sulfhydryl groups (after mild reduction or disulfide bonds) or primary amines. Excess labeling reagent and byproducts are then washed away before recovering the labeled and purified antibody from the gel using a mild imidazole solution. No gel filtration or dialysis is needed. Four kits are available for small (0.1-1 mg) or large (1-10 mg) antibody samples using either amine-directed (NHS ester) or sulfhydryldirected (maleimide) labeling reagents. Highlights: • Fast labeling and puriication – the entire procedure takes only one hour (two hours for sulfhydryl labeling kits) • Easy removal of spent and excess labeling reagent – simply wash away the reaction byproducts – no need for dialysis or gel filtration • No dilution effects – solid-phase method allows initially dilute antibodies to be recovered in a smaller volume after labeling • Optimized protocols – speciic protocols for antibody ensure appropriate level of labeling (2-5 biotins per antibody molecule), minimizing possibility of inactivation caused by overlabeling • Suficient reagents for eight biotin-labeling experiments – Thermo Scientific No-Weigh Single-Dose Microtube Packaging ensures that the biotin reagent is fully active for eight separate experiments H2N SS H2N SS NH2 SS COO– 2+ NH2 COO–Ni + H N Ni2+ Chelate Support 2 S S NH2 N Phosphate Buffered Saline, pH 7.2 H2N N Purified IgG Class Antibody SS O N O O SS SS H2N O O O O N NH2 N H NHS-PEG4-Biotin SS SS O N–C~~~~Biotin H NH2 H NH O O SS COO– 2+ COO–Ni + O Recovered Biotin~~~~C–H S S N N–C~~~~Biotin Ni2+ Chelate Support O NH2 + O N–C~~~~Biotin H Sulfhydryl-directed Labeling (Maleimide-PEG2-Biotin) 0.1-1 mg IgG 1-10 mg IgG Product # 21450 Product # 21440 Product # 21930 Product # 21920 These kits contain our exclusive No-Weigh Single-Dose Microtube Packaging. A single sealed microtube containing 2 mg of reagent is reconstituted for each biotinylation. The exclusive packaging allows access to fresh reagent on-demand for each solid-phase biotinylation reaction. Ordering Information Product # Description Pkg. Size 21440 NHS-PEG Solid-Phase Biotinylation Kit – Pre-Packed Column Kit Biotinylates antibodies and other proteins that bind to the nickel-chelated support provided. A 1 ml column biotinylates 1-10 mg of antibody and can be re-used 10 times. Includes: Immobilized Nickel Chelated Column BupH™ Phosphate Buffered Saline NHS-PEG4-Biotin 4 M Imidazole Stock Solution 21450 O Biotinylated Antibody HN S Summary of Solid-Phase Biotinylation Protocol. Step 1. Immobilize the IgG a. 1 ml Ni-IDA column (for 1-10 mg of IgG) b. Nickel Chelated Disc (for 0.1-1 mg IgG) Step 2. Add the labeling reagent(s) to the immobilized IgG a. NHS-PEG4 Biotin for amine-directed reactions b. TCEP, followed by Maleimide-PEG2-Biotin for sulfhydryl-directed reactions Step 3. Elute the biotinylated IgG with 0.2 M imidazole NHS-PEG Solid-Phase Biotinylation Kit – Mini-Spin Columns Biotinylate antibodies and other proteins that bind to the rehydrated nickel-chelated discs provided. Each disc can biotinylate 100-1,000 µg of antibody. Includes: Nickel Chelated SwellGel® Discs† Mini-Spin Columns Microcentrifuge Tubes (2 ml) BupH Phosphate Buffered Saline NHS-PEG4-Biotin 4 M Imidazole Stock Solution 21920 Maleimide-PEG Solid-Phase Biotinylation Kit – Pre-Packed Column 1 ml 1 pack 8 x 2 mg 5 ml Kit 10 pack 10 pack 30 pack 1 pack 8 x 2 mg 5 ml Kit Reduces and biotinylates IgG class antibodies and other proteins that bind to the nickel-chelated support provided. A 1 ml column biotinylates 1-10 mg of antibody and can be re-used 10 times. Includes: Bond-Breaker® TCEP Solution, Neutral pH 5 ml Immobilized Nickel Chelated Column 1 ml BupH Tris Buffered Saline 1 pack Maleimide-PEG2-Biotin 8 x 2 mg 4 M Imidazole Stock Solution 5 ml 0.2 M Imidazole SS COO– 2+ COO– Ni ||||||||| H2N SS Amine-directed Labeling (NHS-PEG4-Biotin) SS COO– 2+ NH2 COO– Ni ||||||||| O Biotin~~~~C–H S S N N–C~~~~Biotin O H NH2 N SS Antibody Sample Size 21930 Maleimide-PEG Solid-Phase Biotinylation Kit – Mini-Spin Columns Reduces and biotinylates IgG class antibodies and other proteins that bind to the nickel-chelated support provided. Each disc can biotinylate 100-1,000 µg of antibody. Includes: Nickel Chelated SwellGel Discs Bond-Breaker TCEP Solution, neutral pH Mini-Spin Columns Microcentrifuge Tubes (2 ml) BupH Tris Buffered Saline Maleimide-PEG2-Biotin 4 M Imidazole Stock Solution Kit 10 pack 5 ml 10 pack 30 pack 1 pack 8 x 2 mg 5 ml † See patent information. References Mori, Y., et al. (2009). J. Exp. Med. 206, 183-193. Day, P., et al. (2008). J. Virol. 82, 4638-4646. Draghi, M., et al. (2007). J. Immunol. 178, 2688-2698. To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor. 27 Protein Extraction – Cell Surface Proteins Cell Surface Protein Isolation Kit A. Cell Surface Proteins + – Convenient biotinylation and isolation of cell surface proteins for Western blot analysis. R F E – R F E Integrin α5 – + – + R F E R F E R F E Integrin β1 IGF-1Rβ B. Intracellular Proteins + – + – R F E R F E R F E R F E Calnexin Protein isolation is specific to cell surface proteins. Panels are Western blot results for known cell surface proteins (Panel A) and intracellular proteins (Panel B) from HeLa cells tested with the Cell Surface Protein Isolation Kit. Plus symbol (+) denotes results for cells treated with the Sulfo-NHS-SS-Biotin reagent; minus symbol (-) denotes results for cells that were not treated with the biotin reagent but were otherwise carried through the kit procedure. Lanes are no-sample resin-control (R), flow-through (F) and eluted (E) fractions. Presence of target cell surface proteins in the plus-E and minus-F conditions indicate successful isolation with the kit. Presence of intracellular proteins in F condition of both plus and minus conditions indicates that the labeling and purification procedure is specific to cell surface proteins. Ordering Information Product # Description Pkg. Size 89881 Cell Surface Protein Isolation Kit Kit Sufficient reagents and accessories for eight experiments, each involving four T75 flasks of confluent cells. Includes: EZ-Link Sulfo-NHS-SS-Biotin Quenching Solution Lysis Buffer NeutrAvidin Agarose Wash Buffer Dithiothreitol (DTT) BupH Phosphate Buffered Saline BupH Tris Buffered Saline Spin Columns and Accessories References Yang, B., et al. (2009). FASEB J. 23, 503-512. Lee, Y., et al. (2008). Blood. 111, 885-893. Belenkaya, T., et al. (2008). Dev. Cell. 14, 120-131. Quench Reaction 45 40 Transfer cell pellet to 1.5 ml tube 30 25 20 Harvest Cells 15 10 7.5 5.0 Lyse cells 30 minutes on ice N 1D gel gel N Wash gel then elute with SDS-PAGE sample buffer + 50 mM DTT gel N + N protein – SH Perform electrophoresis or other application B SH B S-S-protein Procedure for the Thermo Scientific Cell Surface Protein Isolation Kit. 28 N For more information, or to download product instructions, visit www.thermo.com/pierce 8 x 12 mg vials 16 ml 4.5 ml 2.25 ml 34 ml 8 x 7.7 mg 2 packs 1 pack Isolate biotinylated proteins on NeutrAvidin Agarose Resin 35 30 minutes at 4˚ C R F E Hsp90 Highlights: • Isolates cell surface proteins – reduces complexity of total cellular protein • Eficiently recovers labeled proteins – cleavable biotin allows for nearly 100% recovery of isolated cell surface proteins • Convenience – all reagents are provided in one kit, along with complete instructions for labeling, cell lysis and purification of cell surface membrane proteins • Western blotting applications – proteins recovered in SDS-PAGE buffer are loaded directly onto polyacrylamide gels • Robust system – protocol designed for diverse cell lines, including NIH 3T3, HeLa, C6 and A431 Biotinylate cells + R F E EGFR The Thermo Scientific Cell Surface Protein Isolation Kit is a complete kit for the convenient biotinylation and isolation of mammalian cell surface proteins, specifically targeting cell surface proteins to the exclusion of intracellular proteins. The kit efficiently labels proteins with accessible lysine residues and sufficient extracellular exposure. The isolation procedure uses a cell-impermeable, cleavable biotinyla-tion reagent (Sulfo-NHS-SS-Biotin) to label surface proteins at exposed primary amines. Cells are then harvested and lysed, and the labeled surface proteins are affinity-purified using NeutrAvidin Agarose Resin. The isolated cell surface proteins contain a small, nonreactive tag of the originally labeled primary amines but are no longer biotinylated (biotin remains bound to the resin). R F E NeutrAvidin Biotin-Binding Protein B Biotin Far-Western Blotting Ordering Information The technique of Western blotting has been adapted for use in the search for protein:protein interactions. A far-Western blot uses a tagged protein other than an antibody to probe a membrane. The probe recognizes and binds to proteins on the membrane through protein:protein interactions. Thus a signal generated from a band on the membrane indicates the presence of a protein that interacts with the probe at that apparent molecular weight. This method can be used to search for unknown protein interactions or to confirm putative interactions. The Thermo Scientific Pierce Far-Western Biotinylated Protein:Protein Interaction Kit uses a biotin-tagged protein as the probe for far-Western blotting. Pull-Down Kit for Biotinylated Proteins A pull-down assay is an in vitro method for identifying or confirming protein:protein interactions using a purified and immobilized bait protein. The immobilized bait protein is used as a specific affinity ligand (much as an antibody is used in immunoprecipitation) to capture interacting “prey” proteins from a lysate or other complex mixture. The Thermo Scientific Pierce Pull-Down BiotinylatedProtein:Protein Interaction Kit requires a biotin-tagged protein for the bait and all other reagents are supplied with the kit. Product # Description Pkg. Size 23500 Far-Western Biotinylated-Protein Interaction Kit 10 mini-gels Materials and methods for the discovery, in-gel or on-membrane, of protein interactions using a biotinylated bait protein as the probe. Includes: Streptavidin-HRP Dilution Buffer (10X) Phosphate Buffered Saline 10% Tween®-20 Pierce In-Gel Stable Peroxide Pierce In-Gel Luminol Enhancer Cellophane Exposure Sheets 0.1 mg 50 ml 17 packs 6 x 10 ml ampules 55 ml 55 ml 10 pack Ordering Information Product # Description Pkg. Size 21115 Pull-Down Biotinylated-Protein Interaction Kit Kit Sufficient materials for conducting 25 pull-down assays using a purified and biotinylated protein as the bait. Includes: Immobilized Streptavidin BupH Tris Buffered Saline Biotin Blocking Buffer Wash Buffer (Acetate, pH 5.0) Elution Buffer (pH 2.8) Spin Cup Columns Accessory Pack Collection Tubes and Caps Accessory Pack 1.5 ml settled gel 1 pack (makes 500 ml) 15 ml 100 ml 50 ml 27 columns 200 x 2 ml tubes, graduated To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor. 29 Avidin-Biotin Binding Biotin Biotin, also known as vitamin H, is a small molecule (MW 244.3) that is present in tiny amounts in all living cells. The valeric acid side chain of the biotin molecule can be derivatized to incorporate various reactive groups that are used to attach biotin to other molecules. Once biotin is attached to a molecule, the molecule can be affinity-purified using an immobilized version of any biotin-binding protein. Alternatively, a biotinylated molecule can be immobilized through interaction with a biotin-binding protein, then used to affinity-purify other molecules that specifically interact with it. We offer biotin-labeled antibodies and a number of other biotinylated molecules, as well as a broad selection of biotinylation reagents to label any protein. Valeric Acid Side Chain O H O NH NH HO Biotin S H MW 244.3 Biotin-Binding Proteins Avidin – The extraordinary afinity of avidin for biotin allows biotin-containing molecules in a complex mixture to be discretely bound with avidin. Avidin is a glycoprotein found in the egg white and tissues of birds, reptiles and amphibians. It contains four identical subunits having a combined mass of 67,000–68,000 daltons. Each subunit consists of 128 amino acids and binds one molecule of biotin. The extent of glycosylation on avidin is high; carbohydrate accounts for about 10% of the total mass of the tetramer. Avidin has a basic isoelectric point (pI = 10–10.5) and is stable over a wide range of pH and temperature. Extensive chemical modification has little effect on the activity of avidin, making it especially useful for protein purification. However, because of its carbohydrate content and basic pI, avidin has relatively high nonspecific binding properties. Streptavidin – Another biotin-binding protein is streptavidin, which is isolated from Streptomyces avidinii and has a mass of 75,000 daltons. In contrast to avidin, streptavidin has no carbohydrate and has a mildly acidic pI (5.5). Thermo Scientific Pierce Streptavidin is a recombinant form having a mass of 53,000 daltons and a near-neutral pI. Streptavidin is much less soluble in water than avidin. There are considerable differences in the composition of avidin and streptavidin, but they are remarkably similar in other respects. Streptavidin is also a tetrameric protein, with each subunit binding one molecule of biotin with affinity similar to that of avidin. Guanidinium chloride will dissociate avidin and streptavidin into subunits, but streptavidin is more resistant to dissociation. Streptavidin contains an RYD sequence similar to the RGD sequence that binds cell surface receptors. The RYD sequence can cause background in some applications. NeutrAvidin Protein – We also offer a deglycosylated version of avidin, known as NeutrAvidin Protein, with a mass of approximately 60,000 daltons. As a result of carbohydrate removal, lectin binding is reduced to undetectable levels, yet biotin-binding affinity is retained because the carbohydrate is not necessary for this activity. NeutrAvidin Protein offers the advantages of a near-neutral pI (6.3) to minimize nonspecific adsorption, along with lysine residues that remain available for derivatization or conjugation. NeutrAvidin Protein yields the lowest nonspecific binding among the known biotin-binding proteins due to its near-neutral pI and lack of both carbohydrate and RYD sequence. 30 Strength of Avidin-Biotin Interaction – The avidin-biotin complex is the strongest known noncovalent interaction (Ka = 1015 M-1) between a protein and ligand. The bond formation between biotin and avidin is rapid and, once formed, is unaffected by extremes of pH, temperature, organic solvents and most denaturing agents. These features of avidin – features that are shared by streptavidin and NeutrAvidin Protein – make immobilized forms of the biotin-binding proteins particularly useful for purifying biotin-labeled proteins or other molecules. However, the strength of the interaction and its resistance to dissociation make it difficult to elute bound proteins from an immobilized support. Harsh, denaturing conditions (8 M guanidine•HCl, pH 1.5 or boiling in SDS-sample loading buffer) are required to efficiently dissociate avidin-biotin complexes. Such conditions damage the support irreversibly so that it cannot be reused, and denature the eluted proteins so that they do not maintain any biological activity. Because of these binding and elution properties, purifications based on avidin-biotin affinity are reserved primarily for small-scale procedures involving immediate analysis of the eluted sample by reducing SDS-PAGE or other denaturing method. On the other hand, it is possible to take advantage of the strong avidin-biotin binding properties in immunoprecipitation (IP) and pull-down procedures because the immunoprecipitated “prey” protein can be recovered using elution conditions that will not also elute the biotinylated antibody or “bait” protein. In some situations, it may be most appropriate to use a cleavable biotinylation reagent to label the target molecule so that it may be recovered from its bound state to immobilized avidin by specific cleavage of the spacer arm between biotin and target molecule rather than by elution of biotin from avidin. Monomeric Avidin – Immobilized Monomeric Avidin was developed to allow the purification of fully functional biotinylated proteins. Unlike other biotin-binding proteins that require harsh, denaturing conditions to elute and recover bound molecules, Monomeric Avidin binds reversibly to biotin and allows gentle elution and recovery of biotinylated molecules using a solution of 2 mM biotin to compete for the biotin-binding sites. This makes it possible to harness the avidin-biotin interaction as a purification tool to recover functional proteins and other biological molecules. For more information, or to download product instructions, visit www.thermo.com/pierce Biotin-Binding Products Immobilized Avidin Products Each of the four biotin-binding proteins discussed is available in a variety of immobilized formats. The support resin used for our Immobilized Avidin, Streptavidin and NeutrAvidin Protein is a crosslinked 6%, beaded agarose. Our Immobilized Monomeric Avidin uses a crosslinked 4% beaded agarose. Thermo Scientific UltraLink Biosupport is a durable, polyacrylamide-based resin with a high surface area, large pore volume and low nonspecific binding. It is suitable for pressures up to 100 psi and linear flow rates up to 3,000 cm/hour. A biotin-binding protein immobilized on beaded agarose or our UltraLink® Biosupport can be used for affinity purification in a column or batch method. NeutrAvidin Protein and Streptavidin are also available bound to polystyrene microplates along with a dried blocking buffer. These 96-well plates are offered in transparent, white or black plates to accommodate a variety of assay types. The plates come in two forms – regular and high-binding capacity. The high-binding capacity plates contain more of the immobilized NeutrAvidin Protein or Streptavidin and are ideal for binding large amounts of small, biotin-containing molecule (e.g., a biotinylated peptide). Streptavidin immobilized on MagnaBind Magnetic Beads is an excellent tool for cell-sorting applications. Strong biotin interaction creates a nearly irreversible bond. A Comparison of Biotin-Binding Proteins The strong association between avidin and biotin can be used in the field of affinity separations. By attaching avidin to a solid support, a biotinylated product can be anchored to the same solid support. The attachment is stable over a wide range of pH, salt concentrations and temperatures. To dissociate biotin from avidin, 8 M guanidine•HCl, pH 1.5 or boiling in SDS-PAGE sample buffer must be used. Immobilized avidin can be used in a variety of applications for the affinity purification of biotinylated macromolecules. In one variation, an antibody that has an affinity for a particular antigen is labeled with biotin. Cells containing the antigen are lysed, then incubated with the biotinylated antibody to form a typical antigen/antibody complex. To isolate the antigen, the crude mixture is passed through an immobilized avidin or streptavidin column, which will bind the complex. After appropriate washes, the antigen can be eluted from the column with a low pH elution buffer. The biotinylated antibody is retained by the column. Applications: • Binding biotinylated anti-transferrin for purifying transferrin from serum1 • Binding biotinylated peptides and elution with an SDS/urea solution2 • Hybridization of biotinylated RNA to its complementary DNA and binding to immobilized avidin, with subsequent elution of the single-stranded DNA3 • Puriication of double-stranded DNA4 Ordering Information Product # Description Pkg. Size 20219 Immobilized Avidin 5 ml Immobilized Avidin 5 x 5 ml Immobilized Avidin Columns 5 x 1 ml 20225 Molecular Weight Avidin Streptavidin NeutrAvidin Protein 67 kDa 53 kDa 60 kDa Biotin-binding Sites 4 4 4 Isoelectric Point (pl) 10 6.8–7.5 6.3 Low High Highest Affinity for Biotin (Kd) 10-15 M 10-15 M 10-15 M Nonspecific Binding High Low Lowest Specificity 20362 Support : Crosslinked 6% beaded agarose Capacity: ≥20 µg biotin/ml gel Support and Capacity: Same as above Support and Capacity: Same as above References 1. Wilchek, M. and Bayer, E.A. (1989). Protein Recognition of Immobilized Ligands. Hutchins, T.W., ed. Alan R. Liss, Inc., pp. 83-90. 2. Swack, J.A., et al. (1978). Anal. Biochem. 87, 114-126. 3. Manning, J., et al. (1977). Biochemistry 16, 1364-1370. 4. Pellegrini, M., et al. (1977). Nucleic Acids Res. 4, 2961-2973. Claypool, S.M., et al. (2002). J. Biol. Chem. 277, 28038-28050. Sharma, K.K., et al. (2000). J. Biol. Chem. 275, 3767-3771. Wilchek, M. and Bayer, E.A. (1989). Protein Recognition of Immobilized Ligands. Hutchins, T.W., ed. Alan R. Liss, Inc., pp. 83-90. To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor. 31 Avidin-Biotin Binding Immobilized Streptavidin Products Immobilized NeutrAvidin Products Same high biotin-binding affinity as avidin with low nonspecific binding. Less nonspecific binding produces cleaner results and better yields. Applications: • Puriication of membrane antigens in conjunction with biotinylated monoclonal antibodies1,2 • Cell-surface labeling with biotinylation reagents, followed by precipitation with immobilized streptavidin3 • Puriication of cell-surface glycoproteins using biotinylated Concanavalin A4 • Recovery of single-stranded DNA for dideoxy sequencing5 Ordering Information Product # Description Pkg. Size Streptavidin Agarose Resin 2 ml Streptavidin Agarose Resin 5 ml 20353 Streptavidin Agarose Resin 10 ml 20351 Streptavidin Agarose Columns 5 x 1 ml Streptavidin UltraLink Resin 2 ml 20347 20349 53113 53114 Support: Crosslinked 6% beaded agarose Capacity: 1–3 mg biotinylated BSA/ml resin 15–28 µg biotin/ml resin Support and Capacity: Same as above Support and Capacity: Same as above Support and Capacity: Same as above Support: UltraLink Biosupport Capacity: ≥฀2 mg biotinylated BSA/ml resin ≥฀24 µg biotin/ml resin 5 ml 53116 Streptavidin Plus UltraLink Resin 2 ml 53117 Streptavidin Plus UltraLink Resin 5 ml 20357 High Capacity Streptavidin Agarose Resin 2 ml High Capacity Streptavidin Agarose Resin 5 ml High Capacity Streptavidin Agarose Resin 10 ml 20359 20361 21344 Support: UltraLink Biosupport Capacity: ≥฀4 mg biotinylated BSA/ml resin ≥฀48 µg biotin/ml resin Support and Capacity: Same as above Support: Crosslinked 6% beaded agarose Capacity: > 10 mg biotinylated BSA/ml of resin Support and Capacity: Same as above Support and Capacity: Same as above MagnaBind Streptavidin Beads Support: 1–4 µm, iron oxide particles Capacity: 2 µg biotin/ml beads Applications: • Immunoprecipitation • Purifying proteins that bind to biotinylated ligands • Capturing biotinylated cell-surface proteins1-3 • Purifying biotinylated peptides4 Product # Description Pkg. Size NeutrAvidin Agarose Resin 5 ml NeutrAvidin Agarose Resin 10 ml 53150 NeutrAvidin UltraLink Resin 5 ml 53151 NeutrAvidin Plus UltraLink Resin 5 ml 29202 High Capacity NeutrAvidin Agarose Resin 5 ml 29204 High Capacity NeutrAvidin Agarose Resin 10 ml 29200 29201 5 ml References 1. Gretch, D.R., et al. (1987). Anal. Biochem. 163, 270–277. 2. Updyke, T.V. and Nicolson, G.L. (1984). J. Immunol. Method 73, 83–95. 3. Lisanti, M.P., et al. (1989). J. Cell Biol. 109, 2117–2127. 4. Buckie, J.W. and Cook, G.M. (1986). Anal. Biochem. 156(2), 463–472. 5. Baqui, M., et al. (2003). J. Biol. Chem. 278, 1206–1211. Ellerbroek, S.M., et al. (2001). J. Biol. Chem. 276, 24833–24842. Huh, K-H. and Wenthold, R.J. (1999). J. Biol. Chem. 274, 151–157. Kilic, F., et al. (2000). Proc. Natl. Acad. Sci. USA 97, 3106–3111. Lesa, G.M., et al. (2000). J. Biol. Chem. 275, 2831–2836. Liu, L.A. and Engvall, E. (1999). J. Biol. Chem. 274, 38171–38176. 32 Highlights: • Carbohydrate-free – just like streptavidin, NeutrAvidin BiotinBinding Protein has no carbohydrate, eliminating nonspecific binding problems due to sugars • No interaction with cell surface molecules – absence of the Arg-Tyr-Asp sequence (present in streptavidin), which mimics the universal cell surface recognition sequence present in a variety of molecules, eliminates cross-reactivity of cell surface molecules • Neutral pl – with a pl of 6.3, NeutrAvidin Protein has a pl that is closer to neutrality than avidin or streptavidin, eliminating electrostatic interaction that contributes to nonspecific binding Ordering Information Streptavidin UltraLink Resin Support and Capacity: Same as above When nonspecific binding is a problem in your application, Thermo Scientific Immobilized NeutrAvidin Products are superior alternatives to avidin or streptavidin. NeutrAvidin Biotin-Binding Protein is a modified avidin derivative that combines several key features to provide biotin-binding with exceptionally low nonspecific binding properties. Support: Crosslinked 6% beaded agarose Capacity: > 20 µg or 80 nmol biotin/ml resin (approx. 1–2 mg biotinylated BSA/ml resin) Support and Capacity: Same as above Support: UltraLink Biosupport Capacity: 12–20 µg biotin/ml gel Support: UltraLink Biosupport Capacity: ≥฀30 µg biotin/ml gel Support: Crosslinked 6% beaded agarose Capacity: > 75 µg biotin/ml resin > 8 mg biotinylated BSA/ml resin Support and Capacity: Same as above References 1. Conti, L.R., et al. (2001). J. Biol. Chem. 276, 41270–41278. 2. Daniels, G.M. and Amara, S.G. (1998). Methods Enzymol. 296, 307–318. 3. Liaw, P.C.Y., et al. (2001). J. Biol. Chem. 276, 8364–8370. 4. Oda, Y., et al. (2001). Nature Biotechnology 19, 379–382. Hiller, Y., et al. (1987). Biochem. J. 248, 67–171. Butler, J.E., et al. (1992). J. Immunol. Method 150, 77–90. Murakami, T., et al. (2000). Proc. Natl. Acad. Sci. USA 97(1), 343–348. Cernuda-Morollon, E., et al. (2001). J. Biol. Chem. 276, 35530–35536. Hiller, Y., et al. (1987). Biochem. J. 248, 67–171. Kim, K., et al. (2001). J. Biol. Chem. 276, 40591–40598. Leighton, B.H., et al. (2002). J. Biol. Chem. 277, 29847–29855. Lesa, G.M., et al. (2000). J. Biol. Chem. 275, 2831–2836. Trotti, D., et al. (2001). J. Biol. Chem. 276, 576–582. For more information, or to download product instructions, visit www.thermo.com/pierce Immobilized Monomeric Avidin and Kit Immobilized Iminobiotin and Biotin Ideal affinity support for gentle, reversible binding of biotinylated proteins. Iminobiotin offers mild dissociation conditions at pH 4. When avidin is coupled to a solid support as the subunit monomer, the specificity for biotin is retained, but the affinity for biotin binding substantially decreases (Ka ~ 108 M-1). The Monomeric Avidin Agarose Resin and Kit can be used to bind biotinylated molecules, and the bound material can be competitively eluted using 2 mM biotin in phosphate-buffered saline (PBS). This technique provides the gentlest elution conditions without contamination of the avidin subunits or substantial loss of column-binding capacity. Highlights: • Puriies biotinylated products under mild elution conditions • Can be regenerated and reused at least 10 times • Exhibits little nonspeciic binding (3% or less) Ordering Information H N H N H N S O Immobilized Iminobiotin Iminobiotin is the guanido analog of biotin. The dissociation constant of the avidin-iminobiotin complex is pH-dependent. At pH 9.5-11.0, the avidin-iminobiotin complex will bind tightly. At pH 4, the avidin-iminobiotin complex will dissociate. Because denaturing agents such as 8 M guanidine•HCI or 4 M urea are not used in the purification, an avidin conjugate has a better chance of maintaining its activity during purification. Use immobilized D-Biotin as an “irreversible linkage” to bind streptavidin conjugates. The biotin-streptavidin interaction can withstand extremes in pH, salt and detergents. Ordering Information Product # Description Pkg. Size Product # Description Pkg. Size 20228 Monomeric Avidin Agarose Resin 5 ml 20221 Iminobiotin Agarose Resin 5 ml Biotin Agarose Resin 5 ml 20267 20227 53146 Support: Crosslinked 4% beaded agarose Capacity: ≥฀1.2 mg biotinylated BSA/ml resin Monomeric Avidin Agarose Resin 10 ml Monomeric Avidin Agarose Kit Kit Immobilized Monomeric Avidin UltraLink Resin 5 ml Biotin 1g Support and Capacity: Same as above Support and Capacity: Same as above Includes: 1 x 2 ml Column, Binding and Elution buffers 20218 Support: UltraLink Biosupport Capacity: ≥฀1.2 mg biotinylated BSA/ml resin 29129 NH HN Agarose Bead To break the avidin-biotin interaction, 8 M guanidine•HCl at pH 1.5 or boiling in SDS-PAGE sample buffer is required. These elution methods may result in denaturation of the biotinylated protein and cause irreversible damage to the support. In addition, avidin or streptavidin will be irreversibly denatured and lose the ability to bind subsequent biotinylated samples. NH Support: Crosslinked 6% beaded agarose Spacer: Diaminodipropylamine Capacity: ≥฀1 mg of avidin/ml resin Support: Pierce CDI Support Spacer: Diaminodipropylamine Capacity: ≥฀2 mg of avidin/ml resin References Gitlin, G., et al. (1987). Biochem. J. 242, 923–926. Wood, G.S. and Warnke, R. (1981). J. Histochem. Cytochem. 29, 1196–1204. Hofmann, K., et al. (1980). Proc. Natl. Acad. Sci. USA 77(8), 4666–4668. Gao, C., et al. (1997). Proc. Natl. Acad. Sci. USA 94, 11777–11782. Hofmann, K., et al. (1980). Proc. Natl. Acad. Sci. USA 77, 4666–4668. References Bernstein, E.M., et al. (1999). J. Biol. Chem. 274(2), 889–895. Sims, K.D., et al. (2000). J. Biol. Chem. 275(7), 5228–5237. Ellerbroek, S.M., et al. (2001). J. Biol. Chem. 276, 24833–24842. Glover, B.P. and McHenry, C.S. (2001). Cell 105, 925–934. Horney, M.J., et al. (2001). J. Biol. Chem. 276, 2880–2889. Oda, Y., et al. (2001). Nature Biotechnology 19, 379–382. Schwarzman, A.L., et al. (1999). Proc. Natl. Acad. Sci. USA 96, 7932–7937. Slatin, S.L., et al. (2002). Proc. Natl. Acad. Sci. USA 99, 1286–1291. To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor. 33 Avidin-Biotin Binding MagnaBind Beads for Convenient Affinity Purification Thermo Scientific MagnaBind Magnetic Beads are ideal for solidphase assays such as ELISAs, radioimmunoassays, cell separation and chemiluminescent immunoassays. Magnetic separation offers the following advantages: • Fast, easy separations • Easily scalable • Separation without the need for centrifugation • Easily automated methodology By using affinity chromatographic properties to purify specific molecules from a complex mixture, MagnaBind Beads offer rapid separations, high recovery and specificity. It is possible to isolate single populations of cells, specific proteins and nucleic acids with MagnaBind Technology. MagnaBind Streptavidin is perfect for tightly coupling your biotinylated protein or for capture of biotinylated oligonucleotides. MagnaBind Biotin Beads are available for purification of any biotin-binding molecule. Magnetic separation is a convenient, bench-top procedure for affinity separations of your molecules. 34 Ordering Information Product # Description Pkg. Size MagnaBind Streptavidin 5 ml 21358 MagnaBind Magnet 96-Well Plate Separator 1 21359 MagnaBind Magnet Microcentrifuge Tubes 1 21357 MagnaBind Magnet for 1.5 ml Microcentrifuge Tubes 1 21344 Support: 1-4 µm, iron oxide particles Capacity: 2 µg biotin/ml beads References Chaudhuri, T.K., et al. (2001). Cell 107, 235-246. Ilver, D., et al. (1998). Science 279, 373-377. Fauzi, H., et al. (2005). Nucleic Acid Res. 33, 2595-2602. Singh, R., et al. (2007). Mol. Cancer Ther. 6, 562-569. Su, X., et al. (2006). J. Biol. Chem. 281, 27982-27990. For more information, or to download product instructions, visit www.thermo.com/pierce NeutrAvidin Coated Polystyrene Plates Purified p60c-src Activity Detection with TK Peptide 2 Highlights: • Easy and gentle immobilization of biotin-containing conjugates • Lowest nonspeciic binding properties of all biotin-binding proteins • NeutrAvidin Biotin-Binding Protein has no carbohydrate and an isoelectric point of 6.3 • No denaturing of the protein component of a conjugate upon binding to the plate • Ideal for binding small hydrophilic molecules (e.g., peptides) that typically exhibit poor binding directly to polystyrene • Pre-blocked with your choice of Thermo Scientiic Blocker BSA or SuperBlock Blocking Buffer • Available in 96- and 384-well formats Characteristics of avidin-biotin proteins. Protein Isoelectric Point Contains Carbohydrate Nonspecific Binding Avidin 10–10.5 Yes High Streptavidin 5.5 No Low NeutrAvidin Biotin-Binding Protein 6.3 No Ultralow 1.5 Net Absorbance at 450 nm The high affinity of avidin for biotin, without the nonspecific binding problems. 1.0 0.5 0 0.00 0.05 0.10 0.15 Units Kinase Biotinylated tyrosine kinase peptide 2 was added to Thermo Scientific NeutrAvidin Coated Plates and incubated for 30 minutes. Wells were washed; samples containing p60c-src tyrosine kinase were added to phosphorylate the tyrosine residue on the peptide. Anti-phosphotyrosine monoclonal antibody conjugated to horseradish peroxidase was added. Tyrosine kinase activity was detected by Thermo Scientific 1-Step Turbo TMB Substrate. Kinase activity was quantitated by comparison with a standard curve generated using the phosphorylated form of the same peptide substrate. Reference Singh, Y., et al. (1999). Infect. Immun. 67, 1853–1859. Ordering Information Product # Coating Plate Type Blocking* Binding Capacity† Pkg. Size 15129 Clear, 96-Well SuperBlock BB, 200 µl ~ 15 pmol biotin/well 5 plates NeutrAvidin Protein, 100 µl 15127 NeutrAvidin Protein, 100 µl Clear, 8-Well Strip SuperBlock BB, 200 µl ~ 15 pmol biotin/well 5 plates 15400 NeutrAvidin Protein, 50 µl Clear, 384-Well SuperBlock BB, 100 µl ~ 10 pmol biotin/well 5 plates White, 96-Well SuperBlock BB, 200 µl ~ 15 pmol biotin/well 5 plates 15116 NeutrAvidin Protein, 100 µl 15401 NeutrAvidin Protein, 50 µl White, 384-Well SuperBlock BB, 100 µl ~ 10 pmol biotin/well 5 plates 15117 NeutrAvidin Protein, 100 µl Black, 96-Well SuperBlock BB, 200 µl ~ 15 pmol biotin/well 5 plates 15402 NeutrAvidin Protein, 50 µl Black, 384-Well SuperBlock BB, 100 µl ~ 10 pmol biotin/well 5 plates 15123 NeutrAvidin Protein, 200 µl Clear, 96-Well Blocker BSA, 300 µl > 15 pmol biotin/well 5 plates 15128 NeutrAvidin Protein, 200 µl Clear, 8-Well Strip Blocker BSA, 300 µl > 15 pmol biotin/well 5 plates 15216 NeutrAvidin Protein, 200 µl White, 96-Well Blocker BSA, 300 µl > 15 pmol biotin/well 5 plates 15217 NeutrAvidin Protein, 200 µl Black, 96-Well Blocker BSA, 300 µl > 15 pmol biotin/well 5 plates 15115 Biotin Binding Plate Sample Pack, one each of Product #s 15120, 15121, 15127, 15128 4 plates * BB = Blocking Buffer † Approximate values; plates tested for specific signal:noise and C.V. All coated 96- and 384-well plates are available in bulk quantity with bulk packaging at a discounted price. We can also custom-coat plates using a certain type of plate or a specific supplier’s plate or coat with a specific surface chemistry that is not included in our standard product offering. Please contact our Large-Volume Custom Sales Team at 800-874-3723 or 815-968-0747 for more information. Outside the United States, contact your local branch office or distributor. To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor. 35 Avidin-Biotin Binding 10 NeutrAvidin High Binding Capacity (HBC) Coated Plates We offer researchers a wide variety of avidin-biotin products, including our exclusive NeutrAvidin Coated Plates available in a high binding capacity (HBC) format. NeutrAvidin Protein is a deglycosylated form of avidin with a near-neutral pI that results in less nonspecific binding than that of streptavidin or avidin. Our patent-pending plate-coating technology offers a NeutrAvidin HBC Plate with a wider detection limit than our regular binding capacity plates. The standard curve exhibits greater linearity for detecting small biotinylated molecules such as peptides (see Figure) and oligonucleotides, resulting in greater assay precision. Try Thermo Scientific Pierce NeutrAvidin HBC Coated Plates for binding small biotinylated ligands and see the difference. Highlights: • Unique plate-coating technology – results in high loading of NeutrAvidin Protein per well • Improved sensitivity – less nonspeciic binding for improved signal-to-noise ratios • Broader dynamic range – extends the quantitative range so there’s no need for dilutions • Save time – pre-blocked plates to reduce the number of assay steps • Flexible assay formats – coated plates offered in 96- and 384-well formats and in different colors S/N Ratio Unique technology for improved assay precision. HBC 8 RBC CHC 6 4 2 0 0 5 10 15 Biotinylated Phosphopeptide (pM/well) Comparison of Thermo Scientific NeutrAvidin High Binding Capacity (HBC) Coated Plate, NeutrAvidin Regular Binding Capacity (RBC) Coated Plates and another supplier’s Streptavidin Coated High Binding Capacity Plates (CHC). Plates were incubated with various dilutions of biotinylated, phosphorylated peptide. After washing, the plates were incubated with mouse anti-phosphotyrosine antibody (1:1,000) and then detected using an anti-mouse-FITC conjugate (1:666). The Y-axis is described as the signal-to-noise (S/N) ratio. Ordering Information Product # Coating Plate Type Blocking* Binding Capacity† Pkg. Size 15507 NeutrAvidin Protein, 100 µl Clear, 96-Well SuperBlock BB, 200 µl ~ 60 pmol biotin/well 5 plates 15508 NeutrAvidin Protein, 100 µl Clear, 8-Well Strip SuperBlock BB, 200 µl ~ 60 pmol biotin/well 5 plates 15511 NeutrAvidin Protein, 50 µl Clear, 384-Well SuperBlock BB, 100 µl ~ 35 pmol biotin/well 5 plates 15509 NeutrAvidin Protein, 100 µl White, 96-Well SuperBlock BB, 200 µl ~ 60 pmol biotin/well 5 plates 15512 NeutrAvidin Protein, 50 µl White, 384-Well SuperBlock BB, 100 µl ~ 35 pmol biotin/well 5 plates 15510 NeutrAvidin Protein, 100 µl Black, 96-Well SuperBlock BB, 200 µl ~ 60 pmol biotin/well 5 plates 15513 NeutrAvidin Protein, 50 µl Black, 384-Well SuperBlock BB, 100 µl ~ 35 pmol biotin/well 5 plates * BB = Blocking Buffer † Approximate values; plates tested for specific signal:noise and C.V. 36 For more information, or to download product instructions, visit www.thermo.com/pierce Pierce Streptavidin Coated Polystyrene Plates The specific binding affinity of streptavidin for biotin – in a microplate. Highlights: • Easy and gentle immobilization of biotin-containing conjugates • Low nonspeciic binding • No denaturing of the protein component of a conjugate upon binding • Ideal for binding small biotinylated hydrophilic molecules (e.g., peptides) that typically exhibit poor binding to polystyrene • Pre-blocked with your choice of Blocker BSA or SuperBlock® Blocking Buffer • Available in clear, white and black plates in 12 × 8-well strip, 96-well and 384-well formats References Estrada, G., et al. (1996). Mol. Cell Probes 10, 179–185. Grobler, J.A. et al. (2002). Proc. Nat. Acad. Sci., USA 99, 6661–6666. Ordering Information Product # Coating Plate Type Blocking* Binding Capacity† Pkg. Size 15124 Streptavidin, 100 µl Clear, 96-Well SuperBlock BB, 200 µl ~ 5 pmol biotin/well 5 plates 15126 Streptavidin, 100 µl Clear, 96-Well SuperBlock BB, 200 µl ~ 5 pmol biotin/well 5 x 5 plates Clear, 8-Well Strip SuperBlock BB, 200 µl ~ 5 pmol biotin/well 5 plates 15120 Streptavidin, 100 µl 15122 Streptavidin, 100 µl Clear, 8-Well Strip SuperBlock BB, 200 µl ~ 5 pmol biotin/well 5 x 5 plates 15405 Streptavidin, 50 µl Clear, 384-Well SuperBlock BB, 100 µl ~ 4 pmol biotin/well 5 plates 15118 Streptavidin, 100 µl White, 96-Well SuperBlock BB, 200 µl ~ 5 pmol biotin/well 5 plates 15119 Streptavidin, 100 µl Black, 96-Well SuperBlock BB, 200 µl ~ 5 pmol biotin/well 5 plates 15407 Streptavidin, 50 µl Black, 384-Well SuperBlock BB, 100 µl ~ 4 pmol biotin/well 5 plates 15125 Streptavidin, 200 µl Clear, 96-Well Blocker BSA, 300 µl ~ 10 pmol biotin/well 5 plates 15121 Streptavidin, 200 µl Clear, 8-Well Strip Blocker BSA, 300 µl ~ 10 pmol biotin/well 5 plates 15218 Streptavidin, 200 µl White, 96-Well Blocker BSA, 300 µl ~ 10 pmol biotin/well 5 plates 15219 Streptavidin, 200 µl Black, 96-Well Blocker BSA, 300 µl ~ 10 pmol biotin/well 5 plates 15115 Biotin Binding Plate Sample Pack, one each of Product #s 15120, 15121, 15127, 15128 4 plates * BB = Blocking Buffer † Approximate values; plates tested for specific signal:noise and C.V. To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor. 37 Avidin-Biotin Binding Pierce Streptavidin HBC Coated Plates 180 Thermo Scientific Pierce Streptavidin High Binding Capacity (HBC) Coated Plates are designed for binding biotinylated oligonucleotides and peptides with higher binding efficiency than other commercially available plates. Our proprietary coating technology (patent pending) has created a streptavidin-coated plate with four- to five-times the binding capacity of other suppliers’ plates. Using our Streptavidin HBC Plate can result in an assay with a broader dynamic range and better linearity, leading to improved assay precision (see Figure). Try our Streptavidin HBC Coated Plate and see what has been going undetected in your research. Highlights: • Broader dynamic range – extends the quantitative range so there’s no need for dilutions • Better sensitivity – increased binding capacity allows direct detection of small ligands not observed with regular binding capacity plates • Superior assay precision – standard curve demonstrates greater linearity • Save time – pre-blocked to reduce number of assay steps • Flexible assay formats – offered in 96- and 384-well formats and in different colors S/N Ratio Take advantage of our technology that provides a broader dynamic range. 160 HBC 140 CHC 120 100 80 60 40 20 0 0 5 10 15 20 25 Comparison of Thermo Scientific Pierce Streptavidin High Binding Capacity (HBC) Coated Plate with another commercially available high binding capacity plate (CHC). Plates were incubated with a biotinylated oligonucleotide, washed and probed with a complementary oligonucleotide labeled with fluorescein at various dilutions. The Y-axis is described as the signal-to-noise (S/N) ratio. Ordering Information Product # Coating Plate Type Blocking* Binding Capacity† Pkg. Size 15500 Streptavidin, 100 µl Clear, 96-Well SuperBlock BB, 200 µl ~ 125 pmol biotin/well 5 plates 15501 Streptavidin, 100 µl Clear, 8-Well Strip SuperBlock BB, 200 µl ~ 125 pmol biotin/well 5 plates Clear, 384-Well SuperBlock BB, 100 µl ~ 60 pmol biotin/well 5 plates 15504 Streptavidin, 50 µl 15502 Streptavidin, 100 µl White, 96-Well SuperBlock BB, 200 µl ~ 125 pmol biotin/well 5 plates 15505 Streptavidin, 50 µl White, 384-Well SuperBlock BB, 100 µl ~ 60 pmol biotin/well 5 plates 15503 Streptavidin, 100 µl Black, 96-Well SuperBlock BB, 200 µl ~ 125 pmol biotin/well 5 plates 15506 Streptavidin, 50 µl Black, 384-Well SuperBlock BB, 100 µl ~ 60 pmol biotin/well 5 plates * BB = Blocking Buffer † Approximate values; plates tested for specific signal:noise and C.V. 38 30 Fluoresceinated Oligonucleotide (pM/well) For more information, or to download product instructions, visit www.thermo.com/pierce Protein Immunodetection Thermo Scientific Avidin-based Conjugates Comparison of Thermo Scientific NeutrAvidin Biotin-Binding Protein, Avidin and Streptavidin. The noncovalent, high affinity of biotin for avidin (Ka = 1015 M-1), with four biotin-binding sites per avidin molecule, allows more signal to be concentrated at a detection site. Below are just a few of the applications exploiting the avidin-biotin interaction. • ELISA • Immunohistochemical staining • Western blotting • DNA hybridization assays • Immunoprecipitation • Afinity chromatography • Fluorescent activated cell sorting (FACS) Thermo Scientific NeutrAvidin Products For ultralow nonspecific binding compared to avidin or streptavidin! Achieve better assay results with the low nonspecific binding properties of NeutrAvidin Protein. NeutrAvidin Protein is deglycosylated, so lectin binding is reduced to undetectable levels without losing biotin-binding affinity (Ka = 1015 M-1). NeutrAvidin Protein neutral pI minimize nonspecific adsorption; lysine residues remain available for derivatization or conjugation through its amine-reactive chemistries. The specific activity for biotin-binding is approximately 14 µg/mg of protein. Protein MW pl Carbohydrate NeutrAvidin Biotin-Binding Protein 60 kDa 6.3 No Streptavidin 53 kDa 6.8-7.5 No Avidin 67 kDa 10 Yes Highlights: • Near-neutral pI (6.3) and no glycosylation, unlike avidin • No RYD recognition sequence like streptavidin • Generally lower nonspeciic binding than avidin and streptavidin • Much lower price than streptavidin References Hiller, Y., et al. (1987). Biochem. J. 248, 167-171. Unson, M.D., et al. (1999). J. Clin. Microbiol. 37, 2153-2157. Wojciechowski, M., et al. (1999). Clin. Chem. 45, 1690-1693. Glover, B.P. and McHenry, C.S. (2001). Cell 105, 925-934. Guo, Y., et al. (2001). J. Biol. Chem. 276, 45791-45799. Claypool, S.M., et al. (2002). J. Biol. Chem. 27, 28038-28050. Ordering Information Product # Description Features Pkg. Size 31000 NeutrAvidin Biotin-Binding Protein • pI that has been reduced to a neutral state • Deglycosylated, so lectin binding is reduced to undetectable levels • Can be used as a biotin blocking agent in tissues for histochemistry • 11-17 µg biotin bound/mg NeutrAvidin Protein 10 mg 31007 Maleimide Activated NeutrAvidin Biotin-Binding Protein • Prepare NeutrAvidin conjugates of proteins/peptides • Reacts spontaneously with free sulfhydryls in the pH range of 6.5-7.5 • 4-8 moles maleimide/mole NeutrAvidin Protein 5 mg 31001 NeutrAvidin Horseradish Peroxidase Conjugated • Better signal-to-noise ratio in assay systems • 1-2 moles HRP/mole NeutrAvidin Protein • 3-8 µg biotin bound/mg conjugate 2 mg 31002 NeutrAvidin Alkaline Phosphatase Conjugated • Lower nonspeciic binding than streptavidin conjugates • Better signal-to-noise ratio in assay systems • 3-8 µg biotin bound/mg conjugate 2 mg 31006 NeutrAvidin Fluorescein Conjugated • Fluorescent-labeled NeutrAvidin Biotin-Binding Protein • Absorption: 490 nm; Emission 520 nm • ≥ 2 moles fluorescein/mole NeutrAvidin Protein 5 mg 22831 DyLight 405 NeutrAvidin • Ex/Em: 400 nm and 420 nm 1 mg 22832 DyLight 488 NeutrAvidin • Ex/Em: 493 nm and 518 nm 1 mg 22837 DyLight 549 NeutrAvidin • Ex/Em: 562 nm and 576 nm 1 mg 22842 DyLight 594 NeutrAvidin • Ex/Em: 593 nm and 618 nm 1 mg 22844 DyLight 633 NeutrAvidin • Ex/Em: 638 nm and 658 nm 1 mg 22845 DyLight 649 NeutrAvidin • Ex/Em: 654 nm and 576 nm 1 mg 22848 DyLight 680 NeutrAvidin • Ex/Em: 682 nm and 715 nm 1 mg 22853 DyLight 800 NeutrAvidin • Ex/Em: 770 nm and 794 nm 1 mg To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor. 39 Protein Immunodetection Thermo Scientific Streptavidin Products Wide selection of conjugates for almost any biotin-based assay. Originally isolated from Streptomyces avidinii, streptavidin is a tetrameric biotin-binding protein that we produce and offer in recombinant form. Compared to the native protein, recombinant streptavidin is smaller that the native protein (MW 53kDa) and has a more neutral isoelectric point (pI 6.8-7.5). Streptavidin is carbohydrate-free and much less soluble in water than avidin, resulting in high binding affinity, capacity and specificity for biotinylated molecules. Ordering Information 40 Product # Description Features Applications Pkg. Size 21122 21125 Streptavidin Streptavidin • Lyophilized, stable powder • No carbohydrate • Much less soluble in water than avidin • 13-22 µg biotin bound/mg of protein • Recombinant • Immunoassay reagent when bound to biotinylated 1 mg enzymes or when conjugated to enzymes 5 mg • Blocking protein for biotin-rich tissue sections (use at 0.1% for inhibition of endogenous biotin) • Can be used with biotinylated enzymes (Product # 29339 or 29139) 21120 Hydrazide Activated • Attaches streptavidin to oxidized carbo- • Used to create immunoassay reagents 2 mg hydrate residues on glycoproteins • Localize glycoproteins on blot transfers, followed • ≥ 4 moles hydrazide/mole streptavidin by detection with a biotinylated enzyme 21102 Maleimide Activated • Attaches streptavidin to sulfhydryls 21126 21124 21127 • Used to create immunoassay reagents 1 mg Horseradish Peroxidase Conjugated • 1-2 moles HRP/mole streptavidin Horseradish Peroxidase Conjugated • ≥ 100 peroxidase units/mg conjugate Horseradish Peroxidase Conjugated • Lyophilized, stable powder • 6-9 µg biotin bound/mg conjugate • Histochemistry • Western blotting • Conti, L.R., et al. (2001). J. Biol. Chem. 276, 41270-41278. 1 mg 2 mg 5 mg 21130 21132 21134 High Sensitivity HRP Conjugated High Sensitivity HRP Conjugated High Sensitivity HRP Conjugated • 1 mg/ml • 1 mg/ml • Pre-diluted (10 µg/ml) • ELISA, Western, IHC • ELISA, Western, IHC • ELISA, Western, IHC 0.5 ml 5 ml 1 mg 21324 Alkaline Phosphatase Conjugated • ≥ 3 µg biotin bound/mg conjugate • Histochemistry 1 mg 21323 Alkaline Phosphatase Conjugated • ≥ 100 phosphatase units/mg conjugate • Western blotting • Harriman, G.R., et al. (1999). J. Immunol. 162, 2521-2529. 3 mg 21224 Fluorescein (FITC) Conjugated • Ex/Em: 490 nm and 520 nm • 3-5 moles FITC/mole streptavidin • Histochemical staining • Fluorescence-activated cell sorting (FACS) 1 mg 21724 Rhodamine (TRITC) Conjugated • Excitation: 515-520 nm and 550-555 nm • Emission: 575 nm • 1-3 moles TRITC/mole streptavidin • Histochemical staining • Fluorescence-activated cell sorting (FACS) 1 mg 21624 Texas Red™ Conjugated • Fluorescently labeled streptavidin • Ex/Em: 595 nm and 615 nm • Histochemical staining; can be used in double staining methods • Fluorescence-activated cell sorting (FACS) 1 mg 21627 R-Phycoerythrin Conjugated • Fluorescently labeled streptavidin • Ex/Em: 480, 545 and 565 nm and 578 nm • Histochemical staining • Fluorescence-activated cell sorting (FACS) 1 ml 21629 Allophycocyanin Conjugated • Fluorescently labeled streptavidin • Ex/Em: 650 nm and 660 nm • Histochemical staining • Fluorescence-activated cell sorting (FACS) 0.5 ml 21831 DyLight 405 Streptavidin • Ex/Em: 400 nm and 420 nm • ELISA, Western, FACS, IHC 1 mg 21832 DyLight 488 Streptavidin • Ex/Em: 493 nm and 518 nm • ELISA, Western, FACS, IHC 1 mg 21837 DyLight 549 Streptavidin • Ex/Em: 562 nm and 576 nm • ELISA, Western, FACS, IHC 1 mg 21842 DyLight 594 Streptavidin • Ex/Em: 593 nm and 618 nm • ELISA, Western, FACS, IHC 1 mg 21844 DyLight 633 Streptavidin • Ex/Em: 638 nm and 658 nm • ELISA, Western, FACS, IHC 1 mg 21845 DyLight 649 Streptavidin • Ex/Em: 654 nm and 673 nm • ELISA, Western, FACS, IHC 1 mg 21848 DyLight 680 Streptavidin • Ex/Em: 682 nm and 715 nm • ELISA, Western, FACS, IHC 1 mg 21850 DyLight 750 Streptavidin • Ex/Em: 752 nm and 778 nm • ELISA, Western, FACS, IHC 1 mg 21851 DyLight 800 Streptavidin • Ex/Em: 770 nm and 794 nm • ELISA, Western, FACS, IHC 1 mg For more information, or to download product instructions, visit www.thermo.com/pierce Thermo Scientific Avidin Products Convenient conjugates for assay detection. Avidin is a tetrameric glycoprotein (MW 67kDa) purified from chicken egg white. The highly specific interaction of avidin with biotin makes it a useful tool in designing nonradioactive detection systems. The extraordinary affinity of avidin for biotin (Ka = 1015 M-1) allows biotin-labeled molecules to be detected with excellent sensitivity and specificity. Avidin is more soluble than streptavidin and has an isoelectric point (pI) of 10.5. It is also more economical than streptavidin, and is commonly used in signal amplification systems such as the ABC system. References Chaiet, I. and Wolf, F.J. (1964). Arch. Biochem. Biophys. 106, 1-5. Savage, M.D., et al. (1992). Avidin-Biotin Chemistry: A Handbook. Rockford, Illinois: Pierce Chemical Company. Wilchek, M. and Bayer, E.A. (1983). Anal. Biochem. 171, 1-32. Gitlin, G., et al. (1987). Biochem. J. 242, 923-926. Bruch, R.C. and White, III, H.B. (1982). Biochemistry 21, 5334-5341. Zuk, P.A. and Elferink, L.A. (2000). J. Biol. Chem. 275, 26754-26764. Ordering Information Product # Description Features Applications Pkg. Size 21121 Avidin Avidin • Immunoassay reagent when bound to biotinylated enzymes or when conjugated to enzymes 10 mg 21128 • Hen egg white glycoprotein, affinity-purified, salt-free, lyophilized powder • 11-14 µg biotin bound/mg avidin • Blocking protein for biotin-rich tissue sections (use at 0.1% for inhibition of endogenous biotin) 20 mg • Isoelectric point of 10-10.5 • Stable over a wide range of pH and temperatures 21123 Horseradish Peroxidase Conjugated 29994 Horseradish Peroxidase Conjugated • Puriied using special afinity techniques to eliminate nucleic acids • 1-2 moles HRP/mole avidin • Use in immunohistochemistry where endogenous phosphatase is a problem 2 mg • Western blotting 5 mg • Use for immunohistochemistry where high levels of endogenous peroxidase is a problem 100 units • 5-10 µg biotin bound/mg protein • ≥ 80 peroxidase units/mg protein 21321 Alkaline Phosphatase Conjugated • Homogeneous by SDS-PAGE • Puriied using special afinity techniques to eliminate nucleic acids • ~1 mole alkaline phosphatase/ mole avidin • Western blotting • ELISA • One unit = 1.0 micromole of p-nitrophenol liberated from p-nitrophenylphosphate per minute at 37°C, pH 9.5 21221 Fluorescein (FITC) Conjugated • Fluorescent-labeled avidin • Ex/Em: 490 nm and 520 nm • Fluorescence-activated cell sorting (FACS) 5 mg • Histochemical staining • No free luorescein • ~3.5 moles luorescein/mole avidin 21021 R-Phycoerythrin Conjugated • Fluorescent-labeled avidin • Fluorescence-activated cell sorting (FACS) • Ex/Em: 450-570 nm and 574 nm • Histochemical staining 1 mg To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor. 41 ABC Staining Kits ABC Staining Kits Thermo Scientific Pierce ABC Staining Kits are highly sensitive, have rapid avidin-biotin interactions and produce very low background staining. Highly diluted primary antibodies can be used with Thermo Scientific Pierce Staining Kits, producing comparable stain intensity to other methods that require higher concentrations of antibody. Two types of enzymatic ABC staining kits, alkaline phosphatase and horseradish peroxidase, are available. The kits can come with or without (standard kit) a biotinylated secondary antibody. Kits with a secondary antibody are selected according to the species of primary antibody to be used. For example, if the primary antibody (IgG) is produced in mice, the kit selected to detect this antibody should be the ABC Mouse IgG Kit. To create a sensitive detection system, you need your specific primary antibody, an ABC Mouse Kit with biotinylated antibody, and an enzyme substrate. The standard kit includes only the avidin and biotinylated enzyme and is useful if an ABC Kit is not available for your specific species of primary antibody, or if the primary antibody is already labeled with biotin. The biotinylated antibody can then be used, along with a blocking agent, and used with a standard kit. The ABC Kits contain: • 3 ml of the appropriate blocking serum • 1 ml of the biotinylated afinity-puriied secondary antibody • 2 ml each of the avidin and biotinylated enzyme reagents Our Ultra-Sensitive ABC Peroxidase Staining Kits are more sensitive than the ABC Peroxidase Staining Kits, without exhibiting increased background staining. These kits supply the extra sensitivity needed for localizing antigens present in very small quantities. An expensive primary antibody may be diluted approximately five-fold higher than it could be with the ABC Peroxidase Kit, while producing equal staining intensity. Ordering Information Product # Description Pkg. Size 32052 Kit Includes: Biotinylated Anti-Mouse IgG Antibody Blocking Buffer Avidin Biotinylated HRP 32054 Comparison of the Relative Sensitivities of Three Amplification Methods vs. a Direct ELISA Technique Absorbance at 405 nm Direct ELISA 32020 LAB ELISA 1.50 BRAB ELISA 1.25 Ultra-Sensitive ABC Peroxidase Rabbit IgG Staining Kit Kit Standard Peroxidase Staining Kit Kit Ultra-Sensitive ABC Standard Peroxidase Staining Kit Kit Includes: Biotinylated Anti-Rabbit IgG Antibody Blocking Buffer Avidin Biotinylated HRP 2.00 1.75 Ultra-Sensitive ABC Peroxidase Mouse IgG Staining Kit ABC ELISA Includes: Avidin Biotinylated HRP 1.00 32050 0.75 0.50 Includes: Avidin Biotinylated HRP 0.25 0.00 0 250 500 750 1,000 1,250 1,500 Antigen (ng) 42 For more information, or to download product instructions, visit www.thermo.com/pierce Biotin Conjugates Notes: • Water-soluble • Perfect for diagnostic applications • Absorbance: 492 nm • Color: greenish-yellow Biotin Fluorescein 31.62 Å O HN H NH H H N S O O O O HO N H (+)-Biotinyl-fluorescein MW 732.80 O O O HO Ordering Information Product # Description Pkg. Size 22030 Biotin Fluorescein 5 mg Ordering Information Product # Notes Description Pkg. Size 29130 • Commonly used as a molecular weight marker on SDS-PAGE and in gel permeation • Frequently used as a blocking agent in many immunological techniques • Useful as a control in ELISA, immunoblotting and immunohistochemical studies • Supplied as a lyophilized powder with 8-12 moles of biotin per mole of BSA Biotinylated Bovine Serum Albumin (BSA) 25 mg 29139 • An extremely pure reagent with virtually all enzyme activity retained after biotinylation Biotinylated Horseradish Peroxidase • Offers excellent sensitivity and is recommended for use in sandwich techniques that utilize Thermo Scientific Pierce Avidin, Streptavidin or NeutrAvidin Protein • Unit is defined as amount of Biotinylated-HRP required to form 1 µmole of purpurogallin from pyrogallol in 20 seconds at 20°C • Reconstituted with distilled water to 1 mM citrate, pH 6 29339 • Alkaline phosphatase has been isolated from calf intestine • Biotinylation of this enzyme does not alter its high specific activity • Can be used as a secondary marker in immunohistochemical staining • One unit equals the amount of protein needed to hydrolyze 1.0 µmole of p-nitrophenyl phosphate per minute at 25°C in the following buffer: 0.1 M glycine, 1.0 mM ZnCl2, 1.0 mM MgCl2, 6 mM PNPP, pH 10.4 Biotinylated Alkaline Phosphatase 1 mg 29939 • Used with ONPG for histochemical and immunoblotting applications • Can be used as a detection reagent • One unit = 1 µmole o-nitrophenyl-β-D-galactopyranoside (ONPG) hydrolyzed per minute at 37°C, pH 7.3 Biotinylated β-Galactosidase 100 units 29988 • Useful as a substitute for biotinylated secondary antibodies • Can detect or locate immunoglobulins on cell surface or in tissue • Recombinant form of Protein G, altered to remove the albumin binding sites to reduce nonspecific binding Biotinylated Protein G 0.5 mg 29989 • Useful as a substitute for biotinylated secondary antibodies • Can detect or locate immunoglobulins on cell surface or in tissue cell surface or in tissue Biotinylated Protein A 1 mg 29997 • Binds to the VL region of kappa light chains (human I, III, IV and Mouse I) without interfering with antigen-binding sites • Binds to all classes of IgG (e.g., IgG, IgM, IgA, IgE and IgD) • Does not bind bovine, goat or sheep immunoglobulins • Binds single-chain variable fragments (ScFv) Biotinylated Protein L .5 mg 31826 • Prepared with highly purified IgG • Detect low concentrations of Fc receptors or anti-immunoglobulin antibodies on cells or in tissue • Use with a NeutrAvidin Conjugate to detect receptors for IgG • Supplied in PBS or HBS (10 mM HEPES, 0.15 M NaCl, pH 7.8) containing 0.04% sodium azide Biotinylated Rabbit IgG 5 mg 29129 • Free biotin is used in the elution buffer to purify biotinylated compounds from monomeric avidin columns • Can be used as a reference standard for the HABA dye assay when measuring degree of biotinylation D-Biotin 1g 5 mg Biotinylated Secondary Antibodies We offer more than 40 biotin-labeled secondary antibodies. View a complete selection guide for secondary antibodies in the Products section of our web site at www.thermo.com/pierce To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor. 43 Example Protocols for Biotinylation Example Protocols for Biotinylation Additional protocols can be found in individual product instruction booklets that can be downloaded from our website. The molar ratio of biotinylation reagent to protein is one of the most important parameters to consider when biotin-labeling a protein. This ratio will determine the degree of labeling that is achieved. A high degree of labeling can ensure that each protein molecule contains at least one biotin moiety. However, a low degree of biotinylation ensures minimal modification of the native protein, resulting in maximum retention of biological activity. For large proteins such as antibodies, a modification level of three to five biotins/protein is generally optimal, while small proteins may require slightly less modification. The optimal biotinylation level for any protein can only be determined experimentally. Biotinylating IgG with EZ-Link Sulfo-NHS-Biotin Reagents Preparing stock solutions of any NHS-esters of biotin with the intent of long-term storage is not recommended because hydrolysis occurs very quickly in solution. The container of biotinylation reagent must be brought to room temperature prior to opening, then the proper amount of biotinylation reagent is weighed, dissolved and used immediately. Even those NHS-esters that require organic solvents should be made up immediately before use as these solvents (i.e., DMSO and DMF) are hygroscopic and water absorbed from the air results in hydrolysis of the reagent. These conditions generally result in an immunoglobulin with a modification level of approximately three to five biotins per molecule of IgG. The molar ratio of Sulfo-NHS-Biotin to protein may be adjusted to achieve a particular outcome. Materials required: • Biotinylation reagent such as Sulfo-NHS-LC-Biotin (Product # 21335) • PBS (0.1 M phosphate, 0.15 M NaCl; pH 7.2; or Product # 28372) or other non-amine containing buffer at pH 7.0-8.5 (recommend pH 7.5-8.0) • Device for removal of unreacted biotin such as Thermo Scientiic Slide-A-Lyzer Dialysis Cassette Kits for dialysis of 0.5-3.0 ml samples (Product # 66382) or Thermo Scientific Zeba Spin Desalting Columns, 5K MWCO, for desalting of samples up to 2 ml (Product # 89891) Procedure: 1. Allow the biotinylation reagent to warm completely to room temperature before opening the vial. 2. Dissolve 2-10 mg of IgG in 1 ml PBS. 3. Immediately before use, make a 10 mM stock solution of SulfoNHS-LC-Biotin. 4. Add sufficient volume of the concentrated Sulfo-NHS-LC-Biotin to give a 12-fold molar excess of biotin to a 10 mg/ml IgG solution or a 20-fold molar excess of biotin to a 2 mg/ml IgG solution (See Table 1 for examples). 5. Place the reaction mixture on ice and incubate for two hours. Alternatively, incubate at room temperature for 30 minutes. 6. Use dialysis or gel filtration to remove the unreacted SulfoNHS-LC-Biotin. See instructions provided with preferred buffer exchange product. 7. Store the biotinylated protein under the same condition that is optimal for the non-biotinylated protein. Amount of biotinylation reagent to add to a 1 ml protein sample. 44 Protein MW of Protein mg Protein/ml mMole Protein/ml Fold Molar Excess of Biotin mM Biotin Reagent to Add Volume of 10 mM Biotin Reagent to add Protein A 42,000 10 2 2.4 x 10-4 4.8 x 10-5 12 20 2.9 x 10-3 9.5 x 10-4 290 µl 95 µl BSA 68,000 10 2 1.5 x 10-4 3.0 x 10-5 12 20 1.8 x 10-3 5.9 x 10-4 180 µl 59 µl IgG 150,000 10 2 6.7 x 10-5 1.3 x 10-5 12 20 8.0 x 10-4 2.7 x 10-4 80 µl 27 µl For more information, or to download product instructions, visit www.thermo.com/pierce Troubleshooting guide for biotinylation with NHS-esters. Problem Cause Solution Poor biotinylation No amines available on molecule of interest Protein loses function • Choose biotinylation reagent that targets different group. • Convert sulfhydryl to amine using Aminoethyl-8 (Product # 23010). Inappropriate choice of buffer • Choose non-amine-containing buffer at pH 7-9 (hydrolysis is very rapid at higher pH). Hydrolysis of biotinylation reagent • Allow reagent to come to RT before opening. • Make up fresh biotinylation reagent. Incomplete removal of primary amines • Use biotinylation reagent immediately. • Dialyze or desalt thoroughly into non-amine-containing buffer at pH 7-9. Over-biotinylation • Reduce molar excess of biotinylation reagent. • Reduce time or temperature for biotinylation. • Choose biotinylation reagent that targets different groups. Biotinylating Cell Surface Proteins Selective labeling of proteins located at the cell surface is often desirable in the study of integral membrane proteins. Cell surface biotinylation has emerged as an important tool for studying the expression and regulation of receptors and transporters, differentiation of plasma membrane proteins from those localized to organelle membranes, and distribution of membrane proteins in polarized epithelial cells. The specificity of Sulfo-NHS-esters of biotin for cell surface labeling has been demonstrated in these applications.2, 3 A variety of similar protocols have been used successfully on a wide range of cell types.1,2,3,4,5,6,7 Because these molecules dissolve readily in polar solutions and are charged by the sodium sulfoxide group on the succinimidyl ring, they cannot permeate the cell membrane. As long as the cell is intact, only primary amines exposed on the surface are biotinylated. When internal biotinylation is desired, our NHS-LC-Biotin (Product # 21336), or other non-water-soluble analogs, can be used. Following cell surface biotinylation, it is often necessary to isolate the biotinylated proteins. This is best accomplished using immobilized NeutrAvidin Biotin-Binding Protein, when harsh elution conditions are appropriate, or Monomeric Avidin, when a milder elution is indicated for the recovery of functional protein molecules. Several example protocols for affinity purification of biotinylated molecules are given in the following pages of this booklet. Materials required: • Sulfo-NHS-LC-Biotin (Product # 21335) or other Sulfo-NHS-ester biotinylation reagent • PBS (0.1 M phosphate, 0.15 M NaCl; pH 7.2; or Product # 28372) References 1. Altin, J.G., et al. (1995). Anal. Biochem. 224, 382-389 2. Daniels, G.M. and Amara, S.G. (1998). Methods. Enzymol. 296, 307-318. 3. Huh, K-H. and Wenthold, R.J. (1999). J. Biol. Chem. 274, 151-157. 4. Leighton, B.H., et al. (2002). J. Biol. Chem. 277, 29847-29855. 5. Liu, L.A. and Engvall, E. (1999). J. Biol. Chem. 274, 38171-38176. 6. Schuberth, H.J., et al. (1996). J. Immunol. Methods 189, 89-98. 7. Schwarzman, A.L., et al. (1999). Proc. Natl. Acad. Sci. U.S.A. 96, 7932-7937. Yang, B., et al. (2009). FASEB J. 23, 503-512. Lee, Y., et al. (2008). Blood. 111, 885-893. Belenkaya, T., et al. (2008). Dev. Cell. 14, 120-131. Procedure: 1. Wash cells three times with ice-cold PBS, pH 8.0, to remove any contaminating proteins. 2. Suspend the cells at a concentration of ~25 x 106 cells/ml in PBS, pH 8.0. Note: Other cell concentrations can be used. The concentration of biotinylation reagent can be scaled up or down accordingly, based on cell size, type, etc. 3. Add 1 mg of Sulfo-NHS-LC-Biotin per ml of reaction volume. 4. Incubate at room temperature for 30 minutes. Note: Performing this incubation at 4°C may reduce active internalization of the biotinylation reagent. 5. Wash cells three times with ice-cold PBS + 100 mM glycine to quench any remaining biotinylation reagent. The cell surface proteins are now biotinylated on exposed lysine residues. Thermo Scientific Pierce Cell Surface Protein Isolation Kit is a convenient way to biotin-label and purify mammalian cell-surface proteins. This kit efficiently labels proteins with accessible lysine residues and sufficient extracellular exposure. The kit includes all the reagents necessary for labeling, cell lysis and purification of cell-surface proteins. Ordering Information Product # Description Pkg. Size 89881 Kit Cell Surface Protein Isolation Kit Includes: EZ-Link Sulfo-NHS-SS-Biotin Quenching Solution Lysis Buffer Immobilized NeutrAvidin Gel Wash Buffer Column Accessory Pack No-Weigh Dithiothreitol (DTT) BupH Phosphate Buffered Saline BupH Tris Buffered Saline 8 x 12mg vials 16 ml 4.5 ml 2.25 ml gel slurry 34 ml 8 spin columns 8 x 7.7 mg 2 packs 1 pack To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor. 45 Example Protocols for Biotinylation One-Step Biotinylation and Dialysis in a Slide-A-Lyzer Cassette Procedure: 1. Allow the biotinylation reagent to warm completely to room temperature before opening the vial. Small-volume samples have the inherent problem of sample loss due to handling. This can be an especially important complication if several processing procedures are required. The use of a Slide-A-Lyzer Dialysis Cassette (SAL) can prevent sample loss because more than one reaction can be performed in a single container, thus eliminating transfer steps. The following is a sample protocol for biotinylating a small amount of protein. A similar method can be used for other reactions inside the SAL. 2. Immediately before use, make a 10 mM solution of Sulfo-NHS-LC-Biotin. Materials required: • Sulfo-NHS-LC-Biotin (Product # 21335) or other Sulfo-NHS-ester biotinylation reagent 4. Inject the mixture into a port the SAL, leaving an air bubble for mixing. • PBS (0.1 M phosphate, 0.15 M NaCl; pH 7.2; or Product # 28372) • Slide-A-Lyzer Kit (Product # 66382, includes cassettes, syringes, needles and buoys) • Protein solution (This protocol is written for 1 ml of a 2-10 mg/ml solution. The choice of SAL and amount of biotinylation reagent can be adjusted to suit other protein amounts.) 3. Add biotinylation reagent directly to the protein in PBS. Add sufficient volume of the 10 mM Sulfo-NHS-LC-Biotin to give a 12-fold molar excess of biotin to a 10 mg/ml IgG solution or a 20-fold molar excess of biotin to a 2 mg/ml IgG solution (See Table 1 for examples). Note: Buffers other than PBS can be used, provided they are at pH 7.0-8.5 and do not contain primary amines. 5. Tape the SAL onto a shaking platform and incubate for 30 minutes to 1 hour at room temperature or 2 hours at 4°C, shaking so that the bubble can be seen “mixing” the reagents. 6. Remove SAL from the shaking platform, insert the needle into another port and withdraw the air bubble. 7. Insert the SAL into the groove in the SAL buoy. Place the cassette and buoy in PBS (or other appropriate buffer) and dialyze. Note: The use of 3 x 500 ml of PBS for 2 hours each is sufficient for dialysis. Less time may be sufficient for some systems. Following dialysis, the sample can be concentrated within the same SAL using Slide-A-Lyzer Concentrating Solution (Product # 66526). 8. Insert the needle into any port, withdraw the dialyzed sample and transfer to an appropriate storage container such as a microcentrifuge tube. Biotinylation troubleshooting guide. Problem Cause No Avidin binding Insufficient biotinylation Loss of protein activity 46 Solution • Increase concentration of biotin reagent in coupling reaction. • Increase incubation time of coupling reaction and maintain concentration of biotin reagent. • Do not use Tris or glycine buffers when coupling amine-reactive labeling reagents. Biotin spacer arm is too short • Use long chain (LC or LC-LC) analog of biotinylation reagent. Over-biotinylation • Reduce concentration of biotin reagent in protein activity coupling reaction. • Reduce incubation time of coupling reaction and maintain concentration of biotin reagent. Steric hindrance • Direct biotinylation toward different residues (i.e., switch fromamine-reactive to sulfhydryl-reactive reagent). For more information, or to download product instructions, visit www.thermo.com/pierce Example Protocols for Affinity Purification Based on Avidin-Biotin Binding Introduction Affinity purification makes use of a specific binding interaction that occurs between molecules and that is used extensively for the isolation of biological molecules. A single pass through an affinity column can achieve a 1,000- to 10,000-fold purification of ligand from a crude mixture. From a single affinity purification step, it is possible to isolate a compound in a form pure enough to obtain a single band upon SDS-PAGE analysis. In affinity purification, a ligand is immobilized to a solid support. Once immobilized, it specifically binds its partner under mild buffer conditions (often physiologic conditions such as phosphate buffered saline). After binding to the partner molecule, the support is washed with additional buffer to remove unbound components of the sample. An elution buffer is added. The elution buffer disrupts the interaction between the ligand and its binding partner by pH extremes (low or high), high salt, presence of detergents, chaotropic agents that unfold one or both of the molecules, or the removal of some factor required for the pair to bind. Once released, the binding partner can be recovered from the support using additional elution buffer. The buffer can then be exchanged by dialysis or desalting into a more suitable buffer for storage or downstream analysis. Any molecule that has an interacting partner can be attached to a support and used for affinity purification. The extraordinary affinity of avidin for biotin allows biotin-containing molecules to be discretely bound to immobilized avidin. Once biotin is attached to a molecule, the molecule can be affinity purified using an immobilized version of any biotin-binding protein. Alternatively, a biotinylated molecule can be immobilized through interaction with a biotinbinding protein, then used to affinity purify other molecules that specifically interact with it. Affinity Purification of Biotinylated Molecules (Column Format) Materials required: • Immobilized Avidin, Streptavidin or NeutrAvidin Biotin-Binding Protein. • Biotinylated sample in solution (~3 mg biotinylated protein/ml immobilized biotin-binding protein). • Binding buffer: Phosphate Buffered Saline (0.1 M phosphate, 0.15 M NaCl; pH 7.2; Product # 28372). To reduce nonspecific binding, add 1% of a detergent such as NP-40 to the binding buffer. • Elution buffer: 8 M guanidine•HCl, pH 1.5 (Product # 24115). • Columns: Product # 89896 for resin volumes of 2 ml or less or Product # 89898 for resin volumes of 2-10 ml. Procedure: 1. Pack gel into the column. 2. Equilibrate the column with three column volumes of binding buffer. 3. Add biotinylated sample to the column and allow sample to enter the gel bed. Sequentially replace the bottom and top caps and incubate at room temperature for 30 minutes. Note: If the sample is large enough that the entire amount cannot be added at once, incubate for 10-15 minutes and allow some of the solution to pass through the column. Add more sample and incubate. Do not exceed the binding capacity of the gel. 4. Wash the column with 10 column volumes of binding buffer. 5. Elute the biotinylated molecules with 5-10 column volumes of the elution buffer. Collect the eluate in 0.5-1 ml fractions. Monitor protein content by measuring the absorbance of each fraction at 280 nm. 6. Desalt or dialyze the eluted fractions of interest to put them into a more suitable buffer. To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor. 47 Example Protocols for Affinity Purification Based on Avidin-Biotin Binding Affinity Purification Using a Biotinylated Antibody (Column Format) Immunoprecipitation Using a Biotinylated Antibody (Batch Format) Materials required: • Immobilized Avidin, Streptavidin or NeutrAvidin Biotin-Binding Protein. Materials required: • Immobilized Avidin, Streptavidin or NeutrAvidin Biotin-Binding Protein. • Biotinylated antibody in solution (~3 mg biotinylated antibody/ml immobilized biotin-binding protein). • Biotinylated sample in solution (~3 mg biotinylated protein/ml immobilized biotin- binding protein). • Binding buffer: Phosphate Buffered Saline (0.1 M phosphate, 0.15 M NaCl; pH 7.2; Product # 28372). To reduce nonspecific binding, add 1% of a detergent such as NP-40. • Binding buffer: Phosphate Buffered Saline (0.1 M phosphate, 0.15 M NaCl; pH 7.2; Product # 28372). To reduce nonspecific binding, add 1% of a detergent such as NP-40. • Elution buffer: IgG Elution Buffer (Product # 21004), Gentle Ag/Ab Elution Buffer (Product # 21027) or 0.1 M glycine•HCl, pH 2.8. • Elution buffer: Pierce IgG Elution Buffer (Product # 21004), ImmunoPure Gentle Ag/Ab Elution Buffer (Product # 21027) or 0.1 M glycine•HCl, pH 2.8. • Columns: Product # 89896 for resin volumes of 2 ml or less or Product # 89898 for resin volumes of 2-10 ml. Procedure: 1. Pack gel into the column. 2. Equilibrate the column with three column volumes of binding buffer. 3. Add biotinylated antibody to the column and allow solution to enter the gel bed. Replace the bottom and top caps sequentially and incubate at room temperature for 30 minutes. Note: If the sample is large enough that the entire amount cannot be added at once, incubate for 10-15 minutes and allow some of the solution to pass through the column. Add more antibody and incubate. Do not exceed the binding capacity of the gel. 4. Wash the column with 10 column volumes of binding buffer. 5. Add antigen-containing sample to the column and allow it to enter the gel bed. Replace the bottom and top caps sequentially and incubate at room temperature for 1-2 hours. 6. Wash the column with 10 column volumes of binding buffer. 7. Elute the antigen with 5-10 column volumes elution buffer. Collect the eluate in 0.5-1 ml fractions. If using IgG Elution Buffer or 0.1 M glycine•HCl, pH 2.8, immediately adjust the pH by the adding 1/10 volume of 1 M phosphate, pH 7.5. Monitor protein content by measuring the absorbance of each fraction at 280 nm. • Microcentrifuge tubes and Spin Columns (Product # 69725). Procedure: 1. In a microcentrifuge tube, solubilize antigen in 50 µl of binding buffer and add the biotinylated antibody. Adjust the volume of the sample to 0.2 ml with binding buffer. 2. Incubate the sample overnight at 4°C. 3. Mix the immobilized avidin to ensure an even suspension. Add the appropriate amount of immobilized avidin to the tube containing the antigen/biotinylated antibody mixture. 4. Incubate the sample with mixing for 1 hour at room temperature or 4°C. 5. Wash the avidin-bound complex with 0.5-1.0 ml of binding buffer and centrifuge for 1-2 minutes at approximately 2,500 x g. Remove the supernatant. Repeat this wash procedure at least four times and remove the final wash. 6. Add elution buffer to the gel to recover the bound antigen. If using our IgG Elution Buffer or 0.1 M glycine•HCl, pH 2.8, remove the liquid and immediately adjust the pH by adding a suitable more concentrated buffer such as 1 M Tris, pH 7.5 (100 µl of this buffer to 1 ml of the sample is sufficient). Note: If using Gentle Ag/Ab Elution Buffer, wash column with three column volumes of Tris Buffered Saline before antigen elution. The Gentle Elution Buffer is not compatible with phosphate buffers. 8. Desalt or dialyze the eluted fractions into a buffer suitable for the downstream application. Note: The column with the immobilized biotinylated antibody may be reused to purify more antigen. Wash column with 10 column volumes of binding buffer, add a solution of 0.02% sodium azide and store at 4°C. 48 For more information, or to download product instructions, visit www.thermo.com/pierce Contact Information Belgium and Europe, the Middle East and Africa Distributors Tel: +32 53 85 71 84 France Tel: 0 800 50 82 15 The Netherlands Tel: 076 50 31 880 Germany Tel: 0228 9125650 United Kingdom Tel: 0800 252 185 Switzerland Tel: 0800 56 31 40 1601675 3/09 Printed in the U.S. Email: [email protected] www.thermo.com/perbio United States Tel: 815-968-0747 or 800-874-3723 Customer Assistance E-mail: [email protected] www.thermo.com © 2009 Thermo Fisher Scientific Inc. All rights reserved. These products are supplied for laboratory or manufacturing applications only. Unless indicated otherwise on the inside back cover, all trademarks are property of Thermo Fisher Scientific Inc. and its subsidiaries.