Avidin-Biotin Products TABLE of CONTENTS

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GRASP THE PROTEOME.® Avidin-Biotin Products TABLE OF CONTENTS

EZ-Link® Biotinylation Reagents 2-3

Labeling Reagent Selection Guides 4-5

Amine-Reactive Biotinylation Reagents 6-9

Sulfhydryl-Reactive Biotinylation Reagents 9-11

Carboxyl-Reactive Biotinylation Reagents 12

Carbohydrate/Aldehyde-Reactive Biotinylation Reagents 13

Photoreactive Biotinylation Reagents 14-15

Specialty Biotinylation Reagents 15-16

Avidin-Biotin-Based Kits 16-17

Affinity Supports 18-21

Coated Plates 21-25

NeutrAvidin™ Conjugates 25-26

Avidin Conjugates 26-27

Streptavidin Conjugates 27-28

ABC Staining Kits 28-29

Biotin Conjugates 29-30

Example Protocols for Biotinylation 31-35

Example Protocols for Avidin-Biotin Purification 36-38

References 39

1 EZ-Link® Biotinylation Reagents

The highly specific interaction of avidin with biotin (vitamin H) can be a useful tool in designing nonradioac- tive 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. Pierce offers an extensive line of biotinylation reagents, conjugates and affinity supports that exploit 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). Biotin, a 244 dalton vitamin found in tiny amounts in all living cells, binds with high affinity to avidin, streptavidin and 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. 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. Photoreactive 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. The most frequently used biotinylation reagents, N-hydroxysuccinimide (NHS) esters and N-hydroxysulfo- succinimide (sulfo-NHS) 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. 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 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. 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. Sulfo-NHS-LC-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. 2 Understanding the functional groups available on an antibody is the key to determining a strategy for modification.

■ Primary amine groups (–NH2) are found on lysine sidechains and at the amino terminus of each polypeptide chain. ■ Sulfhydryl groups (–SH) can be generated by reducing disulfide bonds in the hinge region. ■ Carbohydrate residues containing cis-diols can be oxidized to create active aldehydes (–CHO).

Antigen- binding site Light Chains V V S-S L L S-S S-S S-S VH VH CL CL S-S S-S S-S S Fab (Fab')2 NH2 S-S S- S-S CH1 CH1 Hinge NH2 Region S-S Papain S-S Pepsin

S S CarbohydrateCH CH Carbohydrate 2 S S 2 FC

S S NH CH CH Heavy Chains 2 3 S S 3

NH2 Amines on Sulfhydryls created when lysine residues antibody is reduced

3 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

Application Reagent(s) Features/Benefits Biotinylate ELISA or blotting Sulfo-NHS-LC-Biotin (Product Amine-reactive (lysines). Sure to work well for antibody (to detect with # 21335, Kit Product # 21430) nearly any antibody. Very simple and direct. streptavidin-HRP) or affinity-purifying NHS-PEO4-Biotin Amine-reactive (lysines). PEO spacer arm increases antigen (after (Product # 21329, 21330) solubility of biotinylated molecule. immobilizing on streptavidin column) Biotin-LC-Hydrazide (Product Carbohydrate-reactive. Primarily for polyclonal # 21339), plus Sodium meta- antibodies (purified from serum), which usually Periodate (Product # 20504) have required carbohydrate side chains. Biotinylate ELISA or blotting Sulfo-NHS-LC-Biotin (Product Amine-reactive (lysines). Obvious choice for purified (to detect with # 21335, Kit Product # 21430) sizeable proteins. Slight risk of blocking epitopes or protein streptavidin-HRP) or binding sites necessary for other purposes. affinity-purifying receptor or antibody NHS-PEO4-Biotin Amine-reactive (lysines). PEO spacer arm increases (after immobilizing on (Product # 21329, 21330) solubility of biotinylated molecule. streptavidin column) Sulfo-NHS-SS-Biotin Amine-reactive (lysines). Cleavable disulfide spacer (Product # 21331) allows recovery of biotinylated protein from immobilized streptavidin.

Maleimide-PEO2-Biotin Sulfhydryl-reactive. Use only when there is a reason (Product # 21901, 21902) to target sulfhydryls rather than amines. Biotin-LC-Hydrazide (Product Carbohydrate-reactive. Good choice if the protein is known # 21339), plus Sodium meta- to be adequately glycosylated and the carbohydrate is not Periodate (Product # 20504) important for downstream applications. Biotinylate ELISA or blotting Sulfo-NHS-LC-Biotin (Product Amine-reactive. Choose if N-terminus or lysines are purified (to detect with # 21335, Kit Product # 21430) not required for binding reactions. May be difficult peptide streptavidin-HRP) or to remove unreacted reagent. affinity-purifying receptor or antibody NHS-PEO4-Biotin Amine-reactive (lysines). PEO spacer arm increases (after immobilizing on (Product # 21329, 21330) solubility of biotinylated molecule. streptavidin column) Maleimide-PEO2-Biotin Sulfhydryl-reactive. Choose if cysteines are not (Product # 21901, 21902), required for binding reactions (often a terminal plus Immobilized TCEP cysteine is added during peptide synthesis for this (Product # 77712) purpose). Must ensure that peptide is reduced. May be difficult to remove unreacted reagent. Biotin PEO-Amine (Product # Carbohydrate and amine-reactive. Use only if some 21346), plus EDC (Product # peptide polymerization is acceptable along with 22980) and Sulfo-NHS biotinylation. Polymerization may be desirable if it creates (Product # 24510) large enough molecules to recover conjugate by dialysis. Biotinylate PrimarilyApplication for affinity- Reagent(s)Sulfo-NHS-LC-Biotin (Product Features/BenefitsAmine-reactive (lysines). Obvious, most general cell surface purifying or removing # 21335, Kit Product # 21430) choice. Slight risk of block epitopes or binding sites proteins cell surface receptor necessary for other purposes. ligands (after immobilizing NHS-PEO4-Biotin Amine-reactive (lysines). PEO spacer arm increases on streptavidin column) (Product # 21329, 21330) solubility of biotinylated molecule. Sulfo-NHS-SS-Biotin Amine-reactive (lysines). Cleavable disulfide spacer (Product # 21331) allows recovery of biotinylated protein from immobilized NeutrAvidin™ Protein. Cell Surface Biotinylation and Purification Kit (Product # 89888) Biotinylate Capture or detection of Psoralen-PEO-Biotin Provides random labeling of nucleic acid backbone. DNA/RNA oligonucleotides in (Product # 29986) May cause steric hindrance for hybridization. or oligo- ELISA-type applications nucleotides or affinity purification Biotin PEO-Amine (Product # Requires intact 5'- phosphate; provides end-labeled (Capture in streptavidin- 21346), plus EDC (Product # oligo. coated plates or detect 22980) and Sulfo-NHS protein-bound oligo with (Product # 24510) streptavidin-HRP) 4 SELECTION GUIDE 2

Reactive Group Reacts WIth Linkage Formed

NHS-Ester/Sulfo-NHS Ester Primary Amine Amide Bond O O (lysine residue) O

Biotin C O N Biotin C NH Protein O Protein NH2

Maleimide Sulfhydryl Thioether Bond O (cysteine residue – O H not disulfide bonded)

Biotin N Biotin N

O Protein SH O S Protein

Iodoacetyl Thioether Bond O O Biotin C CH2 I Biotin C CH2 S Protein

Pyridyl Disulfide Disulfide Bond

Biotin S S Biotin S S Protein N

Amine Carboxyl Amide Bond (glutamate or aspartate residues) O Biotin NH2 O Biotin NH C Protein Protein C OH (Reaction requires EDC cross-linker)

Hydrazide Oxidized Carbohydrate Hydrazone Bond O O O H Biotin C NH NH2 Protein C H Biotin C N N C Protein H

Azido (Photoactivatable) N NO2 Biotin DNA/RNA, NH Biotin N N3 Protein, Carbohydrates Protein H Ring expansion followed by coupling with primary amine or insertion into double bonds

5 EZ-Link® Biotinylation Reagents The reaction chemistries of NHS- and sulfo-NHS- Label almost anything with biotin using one of our esters are essentially identical: an amide bond is many high-quality biotinylation reagents formed and NHS or sulfo-NHS are leaving groups in the reaction. Because the target for the ester is the Whether you are an expert or are trying biotiny- deprotonated form of the primary amine, the reac- ® lation for the first time, Pierce EZ-Link tion becomes significant at neutral pH values and Reagents and Kits make biotinylation easy. Our above when the amine is able to react with the ester customers depend on our consistent lot-to-lot by nucleophilic attack. Hydrolysis of the NHS-ester quality for reproducible results. Pierce offers is a major competing reaction, and the rate of ® complete EZ-Link Biotinylation Kits with hydrolysis increases with increasing pH. NHS- and buffers, desalting columns and HABA detection sulfo-NHS-esters have a half-life of hydrolysis of reagent to determine the number of biotin 2-4 hours at pH 7. This half-life decreases to just a residues attached to your protein. few minutes at pH 9. There is considerable flexibility in the actual conditions used for conjugating NHS-esters (or sulfo- NHS-esters) to primary amines. Incubation AMINE-REACTIVE BIOTINYLATION REAGENTS temperatures range from 4-37°C, reaction mixture pH values range from 7-9, and incubation The most common target for modifying protein times range from a few minutes to overnight. molecules is the amine group, which is present on Buffers containing amines (such as Tris or the vast majority of proteins due to the abundance glycine) must be avoided because they compete of lysine side chain ε-amines and N-terminal with the reaction. In preparing an NHS-ester α-amines. Based on water solubility, amine-reac- biotin conjugate, a particular set of conditions will tive biotinylation reagents can be divided into two result in a conjugate with optimum properties for groups. NHS-esters of biotin are essentially water- a specific application. The preparation of an opti- insoluble. For reactions in aqueous solution, they mum conjugate is largely dependent on the must first be dissolved in an organic solvent, then degree of incorporation of the label. Because of diluted into the aqueous reaction mixture. The most the variability among proteins, especially the commonly used organic solvents for this purpose number of amines available for conjugation, are DMSO and DMF, which are compatible with conjugation conditions that are optimal for one most proteins at 20% final concentration. The sol- protein may not be optimal for another protein. vent acts as a carrier for the biotinylation reagent, There are several additional features to consider in forming a microemulsion in the aqueous phase and an amine-reactive biotinylation reagent. Because allowing the biotinylation reaction to proceed. The water-insoluble NHS-esters of biotin are membrane- biotin binds in a pocket located 9 Å below the permeable because they do not possess a charged surface of the avidin molecule, the spacer arm group. They may be used for biotinylating internal connected to the biotin is critical. Long spacer arms as well as external components of a cell. reduce steric hindrance and result in enhanced interaction of avidin and biotin. It is sometimes Sulfo-NHS-esters of biotin are soluble up to approx- necessary to remove biotin from a molecule once a imately 10 mM in water. Sulfo-NHS-esters should procedure has been completed, and there are be dissolved in water just before use because they reagents with a cleavable spacer arm that allow this are prone to hydrolysis. The water solubility of procedure. A macromolecule is first reacted with the sulfo-NHS-esters results from the presence of the cleavable biotinylation reagent, then it is used in a sulfonate (–SO3) group on the N -hydroxysuccin- detection or purification system. Finally the biotin imide ring and eliminates the need to dissolve the moiety can be cleaved away, releasing the molecule reagent in an organic solvent. These compounds into solution. Biotin labeling generally reduces the are used for applications that cannot tolerate solubility of a molecule and may result in precipi- organic solvents. Sulfo-NHS-esters of biotin are tation. Unlike sulfo-NHS-esters that lose their also recommended for use as cell surface biotinyla- solubility-enhancing sulfonate group during the tion reagents. Because of the charged sulfonate reaction process, reagents containing a PEO spacer group, these compounds do not penetrate the plas- arm retain their high solubility when bound to a ma membrane; thus biotinylation is restricted to the protein and are an ideal choice when precipitation cell surface. must be overcome.

6 EZ-Link ® Sulfo-NHS-Biotinylation Kit EZ-Link® Sulfo-NHS-LC-Biotin O Note: O O Na S NH ■ Complete kit with an optimized protocol for O O HN H O labeling a protein and determining how much H H N biotin has been attached O N S Pkg. O O Product # Description Size 21420 EZ-Link® Sulfo-NHS- Kit M.W. 556.59 Biotinylation Kit Spacer Arm: 22.4 Å Includes: Note: Sulfo-NHS-Biotin 25 mg ■ Water-soluble for ease of use; ideal labeling Reaction Buffer 1 pack (500 ml) reagent for monoclonal and polyclonal Desalting Column 1 x 10 ml antibodies HABA Dye (10 mM) 1 ml Pkg. Avidin, affinity-purified 10 mg Product # Description Size References: 69, 70 21335 EZ-Link® 100 mg Sulfo-NHS-LC-Biotin References: 4, 5, 26, 38, 39, 40 EZ-Link® Sulfo-NHS-LC- Biotinylation Kit EZ-Link® Sulfo-NHS-LC-LC-Biotin

Note: O O ■ Na -O O H O H HN Complete kit with an optimized protocol for S N N O O N NH labeling a protein and determining how much O H O O S H biotin has been attached Pkg. M.W. 669.75 Product # Description Size Spacer Arm: 30.5 Å 21430 EZ-Link® Sulfo-NHS-LC- Kit Note: Biotinylation Kit ■ Includes: Contains an extra-long spacer arm (30.5 Å) Sulfo-NHS-LC-Biotin 25 mg to reduce steric hindrance Reaction Buffer 1 pack (500 ml) Pkg. Desalting Column 1 x 10 ml Product # Description Size HABA Dye (10 mM) 1 ml 21338 EZ-Link® 50 mg Avidin, affinity-purified 10 mg Sulfo-NHS-LC-LC-Biotin References: 69, 70 References: 44, 54

EZ-Link® Sulfo-NHS-Biotin EZ-Link® Sulfo-NHS-SS-Biotin O O O Na -O HN O O H Na O O O H HN NH S NH O S N N S O O O S H H O H O S O N S O M.W. 443.43 Spacer Arm: 13 Å M.W. 606.69 Spacer Arm: 24.3 Å Note: Pkg. ■ Product # Description Size Disulfide linkage can be cleaved by 21217 EZ-Link® 50 mg reducing agents Sulfo-NHS-Biotin Pkg. References: 4, 11, 16, 37, 44 Product # Description Size 21331 EZ-Link® 100 mg Sulfo-NHS-SS-Biotin For immediate assistance, visit www.piercenet.com References: 13, 26, 62 or call 800-874-3723. Outside the United States, contact your local Perbio Science branch office or distributor.

7 AMINE-REACTIVE BIOTINYLATION REAGENTS (CONT.) NEW!! ™ ® No-Weigh NHS-PEO4-Biotin EZ-Link NHS-LC-LC-Biotin O O O O HN O O O H O HN N O O NH H OOO N N O N N NH O H S O O H S H M.W. 588.67 Spacer Arm: 32 Å M.W. 567.70 Spacer Arm: 30.5 Å Notes: Note: ■ PEO spacer arm increases solubility ■ Contains an extra-long spacer arm (30.5 Å) of conjugates to reduce steric hindrance ■ No-Weigh™ Packaging ensures fresh reagent Pkg. Product # Description Size and eliminates tedious weighing of reagent 21343 EZ-Link ® NHS-LC-LC-Biotin 50 mg Pkg. Product # Description Size ™ 21329 No-Weigh NHS-PEO4-Biotin 8 x 2 mg EZ-Link® NHS-Iminobiotin ® 21330 EZ-Link NHS-PEO4-Biotin 25 mg NH O HN O H NH N ® O EZ-Link NHS-Biotin O H S O HN O M.W. 340.40 O H NH Spacer Arm: 13.5 Å N O H S Notes: O M.W. 341.38 ■ Reversible binding to avidin prevents Spacer Arm: 13 Å protein denaturation during purification ■ Binds to avidin at pH >9.5 and dissociates at pH 4 Pkg. Product # Description Size Pkg. 20217 EZ-Link® NHS-Biotin 100 mg Product # Description Size 21117 EZ-Link® NHS-Iminobiotin 100 mg References: 18, 47 References: 49, 68

EZ-Link® NHS-LC-Biotin O

NH O HN H O H H N N O S O M.W. 454.54 O Spacer Arm: 22.4 Å

Pkg. Product # Description Size 21336 EZ-Link® NHS-LC-Biotin 50 mg References: 9, 53, 61

For immediate assistance, visit www.piercenet.com or call 800-874-3723. Outside the United States, contact your local Perbio Science branch office or distributor.

8 SULFHYDRYL-REACTIVE BIOTINYLATION REAGENTS EZ-Link® PFP-Biotin O The second most common target for modification in biological molecules is free sulfhydryl HN NH H groups, which are found in the form of exposed F H cysteine in a protein or peptide. Sulfhydryl group F O S biotinylation may provide an advantage in some O F F applications; for example, targeting a sulfhydryl group can be used as a method for preserving F 9.6 Å the biological activity of an enzyme when amines are found at the active site. Modification of these (+)-Biotin pentafluorophenyl ester M.W. 410.36 amines may render the enzyme inactive. This complication can be avoided by using derivatives Notes: of biotin that react with sulfhydryls. Because ■ More reactive than NHS esters biotinylation with these reagents must be per- ■ Will react with primary and secondary formed in buffers free of extraneous sulfhydryls, amines at pH 7-9 substances such as 2-mercaptoethanol, dithio- ■ Easier-to-handle and store than NHS esters threitol and mercaptoethylamine must be ■ Great for DNA labeling removed before biotinylation. ■ PEO spacer arm increases solubility Proteins, peptides or other molecules to be of conjugates biotinylated by sulfhydryl-reactive reagents must Pkg. have a free sulfhydryl group (–SH) available; Product # Description Size disulfides will not react with sulfhydryl-specific ® 21218 EZ-Link PFP-Biotin 50 mg biotinylation reagents. If free sulfhydryls are not Reference: 44 available, they can be generated from disulfides by incubation with a reducing agent or from O lysine residues by incubating with modification EZ-Link® TFP-PEO-Biotin NH HN reagents such as 2-Iminothiolane (Traut’s

S Reagent) or SATA. When working with free sulfhydryls, EDTA is generally included in the

H N buffer system for its antioxidative effect. EDTA O chelates trace amounts of metals in solution that O promote disulfide bond formation. Using nitro- O gen-purged buffers is an additional precaution O 4 used to prevent oxidation of the free sulfhydryls. F 0.7 Å H 1 2.6 F N . 7 .W : 3 F M rm Three separate reaction chemistries are employed O O r A F ce O M.W. a710.74 to target sulfhydryl groups for biotinylation. The Sp O Spacer Arm: 32.6 Å most specific method uses reactive maleimide groups, which are 1,000 times more reactive Notes: ■ toward free sulfhydryls than toward amines at Long-chain, water-soluble, polyethylene oxide pH 7. Biotin-BMCC must be dissolved in an (PEO) spacer arm organic solvent, then diluted into an aqueous ■ Tetrafluorophenyl (TFP) ester reacts with reaction mixture. PEO-Maleimide Activated primary and secondary amines at pH 7-9 Biotin, which is water-soluble by virtue of its ■ Glycine and Tris buffers interfere with reaction polyethyleneoxide (PEO) spacer arm, may be ■ PEO spacer arm transfers hydrophilicity to dissolved directly in aqueous solution. The reac- final conjugate tion of maleimide with free thiols is carried out Pkg. at pH 6.5-7.5 because cross-reactivity toward Product # Description Size primary amines can occur at higher pH values. 21219 EZ-Link® TFP-PEO-Biotin 50 mg Hydrolysis of the maleimide group also increases at higher pH values.

9 SULFHYDRYL-REACTIVE BIOTINYLATION REAGENTS (CONT.)

® Iodoacetyl groups are also used to target free EZ-Link Maleimide-PEO2- sulfhydryls. Iodoacetyl-LC-Biotin is not water- Biotin O soluble and must be dissolved in a solvent NH before use in an aqueous reaction mixture. HN H O PEO-Iodoacetyl-Biotin, which is water-soluble by O H H O N S virtue of its PEO spacer arm, may be dissolved N N O H O directly in aqueous solution. The iodoacetyl group O reacts mainly with thiol groups at pH 7.5-8.5, 29.1 Å resulting in a stable thioether bond. Unless pre- cautions are taken, iodoacetyl groups may not (+)-Biotinyl-3-maleimidopropionamidyl-3,6-dioxaoctainediamine be specific for sulfhydryls. The reaction can be M.W. 525.62 directed toward sulfhydryl groups by limiting the molar ratio of Iodoacetyl-Biotin to protein, such Notes: that the concentration of biotin is present at a ■ Water-soluble small excess over the sulfhydryl content. Also, ■ Reacts with sulfhydryl (–SH) groups the reaction pH should be maintained in the at acidic to neutral pH range of 7.5-8.5. Below pH 9, cross-reactivity ■ Avoids potential modification of tyrosine with amine, thioether and imidazole groups is residues that can be associated with minimized. Therefore, maintaining a lower pH Iodoacetyl-Biotin ensures the modification of sulfhydryl groups and not amino groups. If there are no cysteines Pkg. Product # Description Size available, the reaction can be directed at imida- ® zoles by adjusting the pH to 6.9-7.0. However, 21901 EZ-Link 50 mg Maleimide-PEO2-Biotin the incubation time must be increased to a week. 21902 No-Weigh™ Maleimide- 8 x 2 mg Histidyl side chains and amino groups react in PEO2-Biotin Microtubes the deprotonated form and may take part in reac- Reference: 48 tions above pH 5 and pH 7, respectively, although this reaction is much slower than that for sulfhydryls. EZ-Link® Biotin-BMCC O A pyridyldithiol group may also be used to attach HN NH biotin to a free sulfhydryl by disulfide exchange, H O H H resulting in the formation of a mixed disulfide O N N S bond. The reaction of Biotin-HPDP is generally N H O carried out under physiologic conditions O M.W. 533.68 although the process occurs within a wide range Spacer Arm: 32.6 Å of pH conditions and with a variety of buffer components. Pyridyldithiol reactions result in the release of pyridine-2-thione, which cannot Notes: react with free sulfhydryls. The release of this ■ Reacts with sulfhydryl (–SH) groups compound can be measured and the reaction at pH 6.5-7.5 progress monitored by an increasing absorbance ■ Avoids potential modification of tyrosine at 343 nm. Another important feature of this residues that can be associated with method is that the biotin is released by treatment Iodoacetyl-Biotin with reducing agents. ■ Must be dissolved in DMSO or DMF before use Pkg. Product # Description Size 21900 EZ-Link® Biotin-BMCC 50 mg

10 ® EZ-Link® Biotin-HPDP EZ-Link O PEO-Iodoacetyl Biotin NH O HN H O H H NH N S N HN S N S H H O H H O S I N O O N M.W. 539.78 H O Spacer Arm: 29.2 Å 24.68 Å Notes: (+)-Biotinyl-iodoacetamidyl-3,6-dioxaoctanediamine ■ Pyridyldithiol reacts with sulfhydryl (–SH) groups M.W. 542.43 to form a stable disulfide bond between pH 6-9 ■ Pyridine-2-thione leaving group can be used Notes: to measure degree of biotinylation at 343 nm ■ Water-soluble ■ Disulfide bond is cleavable using 50 mM ■ Reacts with sulfhydryl (–SH) groups DTT or 100 mM 2-Mercaptoethanol at basic to neutral pH ■ Must be dissolved in DMSO or DMF before use ■ Coupling reactions occur in the dark Pkg. Product # Description Size Pkg. 21341 EZ-Link® Biotin-HPDP 50 mg Product # Description Size 21334 EZ-Link® 50 mg References: 29, 58 PEO-Iodoacetyl-Biotin References: 34, 44 For immediate assistance, visit www.piercenet.com or call 800-874-3723. Outside the United States, contact EZ-Link® Iodoacetyl-LC-Biotin your local Perbio Science branch office or distributor.

HN NH H O H H I N N S H O M.W. 510.43 Spacer Arm: 27.1 Å

Notes: ■ Reacts with sulfhydryl (–SH) groups at basic to neutral pH ■ Coupling reactions occur in the dark ■ Must be dissolved in DMSO or DMF before use Pkg. Product # Description Size 21333 EZ-Link® Iodoacetyl- 50 mg LC-Biotin References: 60, 66

11 CARBOXYL-REACTIVE BIOTINYLATION REAGENTS Carboxyl groups, in the form of carboxy termini, EZ-Link® Biotin-PEO-Amine aspartate residues or glutamate residues, can also be targeted for biotin labeling using amine-derivatized O biotin molecules. This reaction is mediated by a class NH HN of cross-linkers known as carbodiimides and results H H H in the formation of an amide bond. The reaction with O N H2N O S EDC, the most common carbodiimide cross-linker, is O generally performed in an MES buffer at pH 4.5-5 and requires just minutes to complete. Buffers containing 20.37 Å primary amines (Tris, glycine, etc.) or carboxyls (acetate, citrate, etc.) must be avoided because they (+)-Biotinyl-3,6-dioxaoctanediamine will quench the reaction. Phosphate buffers are also M.W. 374.50 not recommended because they reduce the conjugation efficiency, although this effect can be overcome Notes: ■ by adding more EDC. Use the EDC cross-linker to couple this analog to carboxyl (–COOH) groups Biotin PEO-Amine, Biotin PEO-LC-Amine and ■ Water-soluble 5(Biotinamido)pentylamine are amine-derivatized Pkg. biotin molecules that can be reacted with carboxyl Product # Description Size groups. In addition, any of the hydrazide-derivatized 21346 EZ-Link® Biotin- 50 mg biotin molecules can be reacted with carboxyls under PEO-Amine 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 EZ-Link Biotin-PEO-LC-Amine result in some polymerization of the peptide or protein if the molecule has both carboxyls and primary O amines on its surface. The extent of polymerization NH HN H can be minimized by decreasing the amount of EDC H H H N O N and/or increasing the amount of the biotin reagent 2 O O S used in the reaction. O

22.91 Å

® EZ-Link 5-(Biotinamido)- (+)-Biotinyl-3,6,9-trioxaundecanediamine pentylamine O M.W. 418.55

HN NH H Notes: H H ■ Use the EDC cross-linker to couple this N H2N S analog to carboxyl (–COOH) groups O ■ Water-soluble ■ Longer chain length for reduction of M.W. 328.47 Spacer Arm: 18.9 Å steric hindrance Notes: Pkg. ■ Product # Description Size Use the EDC cross-linker to couple this 21347 EZ-Link® Biotin- 50 mg analog to carboxyl (–COOH) groups PEO-LC-Amine ■ Dissolves in aqueous solutions ■ Coupling with EDC occurs at pH 4-6 Pkg. For immediate assistance, visit www.piercenet.com Product # Description Size or call 800-874-3723. Outside the United States, contact 21345 EZ-Link® 5-(Biotinamido)- 50 mg your local Perbio Science branch office or distributor. pentylamine References: 7, 41 12 CARBOHYDRATE/ALDEHYDE-REACTIVE BIOTINYLATION REAGENTS

Another common target for protein modification amine-containing buffers, are not recommended is the carbohydrate portion of glycoproteins, for use in either the oxidation or biotinylation which can be reacted with hydrazide-derivatives steps because these buffers react with aldehydes, of biotin. Oxidative treatment of glycoproteins quenching their reaction with hydrazides. using 10 mM periodate is used to generate reactive aldehydes from the cis-diols of a variety of ® carbohydrate moieties. EZ-Link Biocytin-Hydrazide O NH OH OH O O HN H NaIO4 O H RCC R' RCH + HCR' H H2N N N S HH H NH2 O Oxidation of a cis-diol to an aldehyde M.W. 386.51 Spacer Arm: 19.7 Å An aldehyde can be reacted specifically with a Notes: hydrazide group at pH 4-6, forming a stable ■ Can be used to label DNA and RNA through hydrazone linkage. Sialic acid residues on glyco- cytosine residues proteins can be specifically oxidized with sodium ■ More water-soluble than Biotin-LC-Hydrazide periodate (NaIO4) under mild conditions. At 1 mM periodate and a temperature of 0°C, oxidation is Pkg. restricted primarily to sialic acid residues. Sialic Product # Description Size 28020 EZ-Link® Biocytin-Hydrazide 25 mg acid residues also can be biotinylated with References: 6, 50, 51 hydrazide derivatives by pretreatment with neu- raminidase to generate galactose groups. The galactose and N-acetylgalactosamine residues EZ-Link® Biotin-Hydrazide on whole cells can be selectively biotinylated with Biotin-Hydrazides by further treatment with O HN galactose oxidase. This enzyme will convert the O H NH primary hydroxyl groups on these sugars to their H2N N H corresponding aldehydes. H S M.W. 258.34 Mild oxidation of an immunoglobulin with sodi- Spacer Arm: 15.7 Å um periodate produces reactive aldehydes from Pkg. the carbohydrate moieties on the Fc portion of Product # Description Size the antibody, which then can be alkylated by a 21339 EZ-Link® Biotin-Hydrazide 100 mg hydrazide. This approach is advantageous for References: 15, 50 use with antibodies because they become biotinylated in a manner that maintains immuno- ® O logical reactivity. This is an ideal method for EZ-Link Biotin-LC-Hydrazide biotinylating polyclonal antibodies because they HN NH H are heavily glycosylated. Monoclonal antibodies O H may be deficient in glycosylation and success H H 2 N N N S with this method will depend on the extent of H glycosylation for a particular antibody. O M.W. 371.50 Temperature, pH of oxidation and the periodate Spacer Arm: 24.7 Å concentration all affect the reaction with Notes: hydrazide derivatives of biotin. Also, because ■ Extended spacer arm reduces steric hindrance glycosylation varies with each protein, optimum ■ Ideal for labeling polyclonal antibodies conditions must be determined for each glyco- Pkg. protein. Each glycoprotein preparation has an Product # Description Size optimum pH for oxidation and for the hydrazide- 21340 EZ-Link® Biotin-LC-Hydrazide 50 mg mediated biotinylation. Tris, or other primary References: 3, 32, 42, 55, 67

13 PHOTOREACTIVE BIOTINYLATION REAGENTS

Proteins, peptides and other molecules that do not EZ-Link® Photoactivatable Biotin contain any of the reactive functional groups men- – tioned previously may be labeled using a photore- N N+ active biotinylation reagent. These reagents O contain an aryl azide group that is chemically inert N O HN H NH until it is exposed to ultraviolet light, causing the N N N H formation of a short-lived, reactive aryl nitrene. N + H H S – CH The half-life of this aryl nitrene intermediate is on O O 3 -4 M.W. 533.65 the order of 10 seconds. The aryl nitrene reacts Spacer Arm: 30 Å rapidly and nonselectively with electron dense sites by addition into double bonds or insertion Notes: into active hydrogen bonds. If the aryl-nitrene fails ■ Covalent attachment occurs in the presence to react, it undergoes ring expansion and becomes of UV light (350-370 nm) reactive toward nucleophiles such as primary ■ Attachment is nonspecific amines and sulfhydryls. Photoactivation and inser- ■ Must be dissolved in DMSO or DMF before use tion into another molecule can be performed in a ■ wide variety of buffer conditions. However, acidic Reaction buffer should be above pH 6 conditions and reducing agents should be avoided Pkg. because they may inactive the aryl azide group. Product # Description Size 29987 EZ-Link® 0.5 mg Photoactivatable Biotin, Biotin-LC-ASA and Photoactivatable Biotin Psoralen-PEO-Biotin each contain a photoactivat- References: 36, 59 able group. When exposed to UV light, they become activated and insert nonspecifically into nearby molecules. These reagents may be used to EZ-Link® Psoralen-PEO-Biotin label proteins and peptides, but they are also useful in labeling DNA, RNA and other molecules that do Great for labeling probes not contain any readily reactive functional groups. 36.86 Å O O Psoralen-PEO-Biotin contains a reactive psoralen O O O HN O O O H NH group and is designed to efficiently label nucleic N O N H H S acids. The psoralen group intercalates into the 9.24 Å O H helix of DNA, allowing it to label efficiently and M.W. 688.79 selectively. The psoralen can also stack along with the bases of single-stranded DNA or RNA increasing labeling efficiency and selectivity. Upon pho- Notes: toactivation, psoralen forms a cross-link with the ■ Water-soluble 5,6 double bond of pyrimidine bases and its pres- ■ Much higher coupling yield than achieved ence does not interfere with hybridization. with photoactivatable biotin ■ Psoralen moiety reacts through a cyclo-addition mechanism with the 5,6 double bond in thymine- and other pyrimidine-containing bases For immediate assistance, visit www.piercenet.com ■ Covalent attachment occurs in the presence or call 800-874-3723. Outside the United States, contact of UV light (>350 nm) for 10-30 minutes your local Perbio Science branch office or distributor. ■ Great for biotinylating DNA/RNA probes ■ Ultrahigh sensitivity relative to radiolabeled probes (<100 femtograms) ■ Doesn’t interfere with hybridization Pkg. Product # Description Size 29986 EZ-Link® 5 mg Psoralen-PEO-Biotin References: 10, 64

14 EZ-Link® Biotin-LC-ASA O Label Transfer with Sulfo-SBED HN NH OH O H H H The label transfer technique is gaining popularity N N S –N as a valuable strategy for identifying relevant pro- H O +N N tein interactions. A label transfer reagent consists M.W. 503.62 of a reactive group that can be bound to a protein Spacer Arm: 29.9 Å of interest, a second reactive group that can be Notes: bound to another protein that interacts with the ■ Iodinatable and photoactivatable protein of interest, a cleavable spacer arm, and a ■ Useful for biotinylation of nucleic acids label that can be used to purify or identify the labeled protein. One advantage of the label trans- Pkg. Product # Description Size fer technique over other in vitro methods for 29982 EZ-Link® Biotin-LC-ASA 2 mg 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 immunopre- SPECIALTY BIOTINYLATION REAGENTS cipitation or pull-down methods because of the number of washing steps and the time involved. O EZ-Link® Biocytin ProFound™ Label Transfer Sulfo-SBED contains HN NH an amine-reactive NHS-ester, a photoreactive H O H group, a cleavable disulfide linkage, and a biotin H label. Available amine groups on a purified pro- N HO S tein are first reacted with the NHS-ester of Sulfo- NH2 O SBED. The labeled protein is then incubated with M.W. 372.48 either a pure preparation of a binding partner or Spacer Arm: 20.1 Å with a complex mixture such as a lysate to search Note: for a binding partner. Complexes form through ■ Useful for synthesizing long-chain protein:protein interactions and the sample is biotinylation reagents exposed to UV light in the 300-366 nm range. Pkg. The photoreactive moiety inserts itself into a Product # Description Size nearby bond trapping the interacting protein in a 28022 EZ-Link® Biocytin 100 mg covalent complex. By reducing the disulfide link- Reference: 5 age, the label can be transferred to the interacting protein. Then the protein can be purified or EZ-Link® PEO-Biotin Dimer detected using the biotin label. O O The potential applications for Sulfo-SBED can be HN NH HN NH greatly expanded with minor variations on this H H H H H H general scheme. Since the first availability of this N O O N S O O O S reagent in 1994, the literature has grown rapidly O O on its use in the study of protein interactions. 43.36 Å Applications include: (+)-Biotinyl-hexaethyleneglycol dimer M.W. 732.95 ■ Searching for putative binding partners Notes: ■ Interaction mapping ■ Long spacer arm biotin dimer for ■ Study of complex assembly mechanisms cross-linking avidin molecules ■ Defining docking site and cofactor ■ Can be used to increase signal in requirements of interactions assays using the avidin-biotin system ■ ■ Water-soluble Determining binding constants ■ Studying refolding interactions Pkg. Product # Description Size ■ Detecting low abundance receptors 22020 EZ-Link® PEO-Biotin Dimer 50 mg ■ Evaluating drug-receptor interactions 15 SPECIALTY BIOTINYLATION REAGENTS (CONT.) AVIDIN-BIOTIN-BASED KITS

ProFound™ Label Transfer EZ™ Biotin Quantitation Kit Sulfo-SBED Protein:Protein Determine the molar ratio of biotin incorporated Interaction Reagent into a protein using the HABA-Avidin method. The HABA dye binds to avidin to produce a yellow-orange-colored complex that absorbs at N– 21.2 Å 500 nm. Biotin, present in solution with this N+ O avidin-HABA complex, will displace the HABA dye N NH and cause the absorbance to decrease. The HN O amount of biotin present can be calculated H HN N directly from the decreased absorbance at 500 S nm. The EZ™ Biotin Quantitation Kit contains pre- O HN O measured doses of the Avidin-HABA mixture and 14.3 Å Biotinylated BSA positive control to simplify reagent preparation and minimize the amount of S 24.7 Å S 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 O O the Pierce Biotechnology web site. ImmunoPure® N O O HABA, Avidin and Biotinylated BSA are also available separately in larger quantities. O Sulfo-SBED S M.W. 879.98 O Spacer Arms Na+O– [(HABA ):Avidin] NHS ester Phenyl azide 14.3 Å 4 NHS ester Biotin 24.7 Å Phenyl azide Biotin 21.2 Å Absorbance ein 500 nm

Notes: [Protein-Biotinn:Avidin] ■ Photoreactive aryl nitrene group couples to moles biotin protein through C-H bonds when activated at 300-350 nm Pkg. ■ Sulfo-NHS ester reacts with amines to form Product # Description Size stable amide bonds at pH 7-9 28005 EZ™ Biotin 24 assays ■ Quantitation Kit Spacer arm contains a biotin group to allow 28010 HABA 10 g recovery of conjugate on immobilized 28050 HABA 50 g avidin column (2-[4´-Hydroxyazobenzene]- ■ Cleavable disulfide bond in the spacer arm benzoic acid) 21121 ImmunoPure® Avidin 10 mg allows molecules to be detached from each other 29130 ImmunoPure® 25 mg Biotinylated BSA Pkg. Product # Description Size 33033 Profound™ Label 10 mg Transfer Sulfo-SBED Protein:Protein Interaction Reagent* References: 1, 14, 20, 25, 27, 28, 30 35, 43, 44, 45, 56, 63 *Protected by U.S. Patent # 5,532,379.

16 Far-Western Blotting Pull-Down Kit for Biotinylated The technique of Western blotting has been Proteins adapted for use in the search for protein:protein A pull-down assay is an in vitro method for identify- interactions. A far-Western blot uses a tagged ing or confirming protein:protein interactions using protein other than an antibody to probe a mem- a purified and immobilized bait protein. The immo- brane. The probe recognizes and binds to pro- bilized bait protein is used as a specific affinity lig- teins on the membrane through protein:protein and (much as an antibody is used in interactions. Thus a signal generated from a band immunoprecipitation) to capture interacting “prey” on the membrane indicates the presence of a proteins from a lysate or other complex mixture. The protein that interacts with the probe at that appar- ProFound™ Pull-Down Biotinylated-Protein:Protein ent molecular weight. This method can be used Interaction Kit requires a biotin-tagged protein for to search for unknown protein interactions or to the bait and all other reagents are supplied with the confirm putative interactions. The ProFound™ Far- kit. Western Biotinylated Protein:Protein Interaction Kit uses a biotin-tagged protein as the probe for far-Western blotting. Pkg. Product # Description Size 21115 ProFound™ Pull-Down Kit Pkg. Biotinylated-Protein: Product # Description Size Protein Interaction Kit 23500 ProFound™ Far-Western 10 mini-gels Sufficient materials for conducting Biotinylated-Protein:Protein 25 pull-down assays using a purified and Interaction Kit biotinylated protein as the bait. Materials and methods for the discovery, Includes: in-gel or on-membrane, of protein Immobilized Streptavidin 1.5 ml interactions using a biotinylated settled gel bait protein as the probe. BupH™ Tris Buffered Saline 1 pack Includes: (makes 500 ml) Streptavidin-HRP 0.1 mg Biotin Blocking Buffer 15 ml Dilution Buffer (10X) 50 ml Wash Buffer(Acetate, pH 5.0) 100 ml Phosphate Buffered Saline 17 packs Elution Buffer (pH 2.8) 50 ml 10% Tween®-20 6 x 10 ml Handee™ Spin Cup Columns 27 columns ampules Accessory Pack UnBlot® Stable Peroxide 55 ml Collection Tubes and Caps 200 x 2 ml tubes, UnBlot® Luminol Enhancer 55 ml Accessory Pack graduated Cellophane Exposure Sheets 10 pack

For immediate assistance, visit www.piercenet.com or call 800-874-3723. Outside the United States, contact your local Perbio Science branch office or distributor.

17 AFFINITY SUPPORTS

Immobilized Avidin, NeutrAvidin™, cific binding of among the known biotin binding pro- Streptavidin and Monomeric Avidin teins. Biotin-Binding Proteins The avidin-biotin complex is the strongest known noncovalent interaction (Ka = 1015 M-1) between a Avidin is a glycoprotein found in the egg white and protein and ligand. The bond formation between tissues of birds, reptiles and amphibia. It contains biotin and avidin is very rapid and, once formed, is four identical subunits having a combined mass of unaffected by extremes of pH, temperature, organic 67,000-68,000 daltons. Each subunit consists of solvents and other denaturing agents. These features 128 amino acids and binds one molecule of biotin. make immobilized forms of avidin particularly useful The extent of glycosylation is very high. for purifying biotin-labeled proteins or other mole- Carbohydrate accounts for about 10% of the total cules. However, the strength of the interaction and its mass of avidin. Avidin has a basic isoelectric point resistance to dissociation make it difficult to elute (pI) of 10-10.5 and is stable over a wide range of pH bound proteins from an immobilized support. Harsh, and temperature. Extensive chemical modification denaturing conditions (8 M guanidine-HCl, pH 1.5 or has little effect on the activity of avidin, making it boiling SDS-sample loading buffer) are required to especially useful for protein purification. Because of efficiently dissociate avidin:biotin complexes. Such its carbohydrate content and basic pI, avidin has rel- conditions damage the support irreversibly so that it atively high nonspecific binding. cannot be reused and denature the eluted proteins, Streptavidin is another biotin-binding protein that is making them lose any biological activity. isolated from Streptomyces avidinii and has a mass of 60,000 daltons. There are considerable differ- Pierce has developed Monomeric Avidin, which also ences in the composition of avidin and streptavidin, binds specifically to biotin, to allow the purification but they are remarkably similar in other respects. of fully functional biotinylated proteins. Unlike other Streptavidin is also a tetrameric protein, with each biotin-binding proteins that require harsh, denaturing subunit binding one molecule of biotin with a similar conditions to elute and recover bound molecules, affinity to that of avidin. Guanidinium chloride will Monomeric Avidin binds reversibly to biotin and dissociate avidin and streptavidin into subunits, but allows gentle elution and recovery of biotinylated streptavidin is more resistant to dissociation. molecules using a solution of 2 mM free biotin. This Streptavidin is much less soluble in water than makes it possible to harness the avidin–biotin inter- avidin. In contrast to avidin, streptavidin has no action as a purification tool to recover functional carbohydrate and has a mildly acidic pI of 5. Pierce proteins and other biological molecules. products use a recombinant form of streptavidin Each of the four biotin-binding proteins discussed with a mass of 53,000 daltons and a near-neutral pI. above is available in a variety of immobilized formats. Because streptavidin lacks any carbohydrate modifi- The support used for ImmunoPure® Immobilized cation and has a near-neutral pI, it has the advan- Avidin, Streptavidin and NeutrAvidin™ Protein is a 6% tage of much lower nonspecific binding. cross-linked, beaded agarose resin. ImmunoPure® Pierce also offers a deglycosylated version of avidin, Immobilized Monomeric Avidin uses a 4% cross- known as NeutrAvidin™ Biotin Binding Protein, with linked, beaded agarose resin. The UltraLink® Resin a mass of approximately 60,000 daltons. As a result is a durable, polyacrylamide-based support with a of carbohydrate removal, lectin binding is reduced high surface area, large pore volume and low non- to undetectable levels, yet biotin-binding affinity is specific binding. It is suitable for pressures up to retained because the carbohydrate is not necessary 100 psi and linear flow rates up to 3,000 cm/hour. for this activity. NeutrAvidin™ Protein offers the Biotin-binding proteins immobilized on agarose or advantages of a near-neutral pI (6.3) to minimize UltraLink® Beads may be used for affinity purifica- nonspecific adsorption, along with lysine residues tion in a column or batch method. NeutrAvidin™ that remain available for derivatization or conjuga- Protein and Streptavidin are also available bound to tion. NeutrAvidin™ Protein yields the lowest nonspe- polystyrene microplates along with a dried blocking

Isoelectric Contains Nonspecific RYD Protein Point Carbohydrate Binding M.W. Sequence Avidin 10-10.5 Yes High 67 kD No Streptavidin 6.8-7.5 No Low 53 kD Yes NeutrAvidin™ 6.3 No Ultra low 60 kD No Biotin-Binding Protein

18 buffer. These 96-well plates are offered in trans- Immobilized Avidin parent, white or black plates to accommodate a variety of assay types. The plates come in two Notes: forms – regular and high-binding capacity. The ■ Stable over a wide pH and temperature range high-binding capacity plates contain more of the ■ High pI and carbohydrate content ™ immobilized NeutrAvidin Protein or Streptavidin contribute to nonspecific binding and are ideal for binding large amounts of a small, biotin-containing molecule (e.g., a biotinylated Pkg. ™ Product # Description Size peptide). Streptavidin immobilized on MagnaBind 20219 Immobilized Avidin 5 ml Magnetic beads is an excellent tool for cell sorting Support: Cross-linked 6% beaded agarose applications. Capacity: ≥20 µg biotin/ml gel 20225 Immobilized Avidin 5 x 5 ml Support and Capacity: Same as above ™ 1.) Affinity Purification: Monomeric Avidin-Biotin Binding 20362 AffinityPak Immobilized 5 x 1 ml Avidin Columns Support and Capacity: Same as above References: 11, 56, 65

B B Immobilized Streptavidin B B Notes: ■ Less nonspecific binding than avidin ■ Lower pI and no carbohydrate; exhibits some B nonspecific binding to cell adhesion proteins

B Pkg. 2.) ELISA Development: Binding of Biotinylated Biomolecules Product # Description Size to Streptavidin-coated Polystyrene Plates 20347 Immobilized Streptavidin 2 ml Streptavidin coated on walls Support: Cross-linked 6% beaded agarose and bottom of microplate well. Capacity: 15-28 µg biotin/ml gel 1-3 mg biotinylated BSA/ml gel

n B R 20349 Immobilized Streptavidin 5 ml Support and Capacity: Same as above ™ Biotinylated Conjugates of: 20351 AffinityPak 5 x 1 ml • Antigens • Haptens Immobilized Streptavidin Columns • Antibodies • Lectins Support and Capacity: Same as above • Carbohydrates • Peptides 53113 UltraLink® 2 ml • Cells • Proteins R Immobilized Streptavidin • DNA • Receptors ® • Enzymes Support: UltraLink Biosupport Capacity: ≥2 mg biotinylated BSA/ml gel B Biotinylated conjugates bound to ≥24 µg biotin/ml gel streptavidin on the surface of the ® microplate well can be properly oriented 53114 UltraLink 5 ml n for further assay development. Immobilized Streptavidin Support: UltraLink® Biosupport Capacity: ≥2 mg biotinylated BSA/ml gel 3.) Immunoprecipitation: Avidin-Biotin Purification of Protein ≥24 µg biotin/ml gel 53116 UltraLink® 2 ml Immobilized Streptavidin Plus Capacity: ≥4 mg biotinylated BSA/ml gel B ≥48 µg biotin/ml gel ® A B 53117 UltraLink 5 ml Immobilized Streptavidin Plus Capacity: ≥4 mg biotinylated BSA/ml gel ≥48 µg biotin/ml gel References: 5, 16, 26, 33, 38, 40

For immediate assistance, visit www.piercenet.com or call 800-874-3723. Outside the United States, contact your local Perbio Science branch office or distributor.

19 AFFINITY SUPPORTS (CONT.)

Immobilized Monomeric Avidin Pierce developed Immobilized Monomeric Avidin to reversibly bind biotin and make an ideal affinity B support for purifying biotinylated molecules. AB Notes: ■ Retains specificity of avidin-biotin interaction, but with lower binding affinity ■ Recovers biotinylated molecules with Avidin-Biotin Purification of Protein gentle elution conditions using free biotin, instead of the 8 M guanidine•HCl normally required for immobilized streptavidin ™ Immobilized NeutrAvidin purification Biotin-Binding Protein ■ Low nonspecific binding Notes: Pkg. ■ Near-neutral pI, deglycosylated and no Product # Description Size nonspecific binding to cell adhesion protein 20228 Immobilized Monomeric 5 ml ■ Avidin Exceptionally low nonspecific binding – Support: Cross-linked 4% beaded agarose even less than streptavidin Capacity: ≥1.2 mg biotinylated BSA/ml gel ■ High-affinity biotin binding 20227 Immobilized Monomeric Kit Avidin Kit Support and Capacity: Same as above Pkg. Includes: 1 x 2 ml Column, Product # Description Size Binding and Elution buffers ® 29200 Immobilized 5 ml 53146 UltraLink 5 ml NeutrAvidin™ Protein Immobilized Monomeric Support: Cross-linked 6% beaded agarose Avidin ® Capacity: ≥20 µg biotin/ml gel Support: UltraLink Biosupport ≥ 53150 UltraLink® 5 ml Capacity: 1.2 mg biotinylated BSA/ml gel Immobilized NeutrAvidin™ Protein 29129 Biotin 1 g ® Support: UltraLink Biosupport References: 16, 21, 25, 48, 54, 58 Capacity: 12-20 µg biotin/ml gel 53151 UltraLink® Immobilized 5 ml NeutrAvidin™ Protein Plus Support: UltraLink® Biosupport Immobilized Iminobiotin Capacity: ≥30 µg biotin/ml gel References: 7, 12, 13, 23, 34, 37, 38, 39, 48, 62 Notes: ■ Dissociation of avidin-iminobiotin complex is pH-dependent ■ Dissociation occurs at pH 4 For immediate assistance, visit www.piercenet.com or call 800-874-3723. Outside the United States, contact Pkg. your local Perbio Science branch office or distributor. Product # Description Size 20221 Immobilized 5 ml Iminobiotin on Agarose Support: Cross-linked 6% beaded agarose Capacity: ≥1 mg avidin/ml gel References: 19, 24

20 COATED PLATES

Immobilized Biotin Reacti-Bind™ NeutrAvidin™ Note: Coated Plates ■ Features high affinity for avidin The high affinity of avidin for biotin, without Pkg. the nonspecific binding Product # Description Size 20218 Immobilized Biotin 5 ml Notes: Support: Reacti-Gel® (6X) Support ■ Easy and gentle immobilization of biotin- ≥ Capacity: 2 mg avidin/ml gel containing conjugates ■ Lowest nonspecific binding properties of all biotin-binding proteins ™ MagnaBind Beads for ■ NeutrAvidin™ Biotin-Binding Protein has no Convenient Affinity Purification carbohydrate and an isoelectric point of 6.3 MagnaBind™ Magnetic Beads are ideal for solid- ■ No denaturing of the protein component phase assays such as ELISAs, radioimmunoas- of a conjugate on binding says, cell separation and chemiluminescent ■ Pre-blocked with your choice of Blocker™ ® immunoassays. BSA or SuperBlock Blocking Buffer ■ Available in 96-well and 384-well formats Magnetic separation offers the Reference: 57 following advantages: ■ Fast, easy separations ■ Easily scalable ■ Separation without the ■ Easily automated Purified p60 c-src Activity Detection with TK Peptide 2 need for centrifugation methodology 1.5 By using affinity chromatographic properties to purify specific molecules from a complex mixture, ™ MagnaBind Beads offer rapid separations, high 1.0 recovery and specificity. It is possible to isolate single populations of cells, specific proteins and ™ nucleic acids with MagnaBind Technology. 0.5 MagnaBind™ Streptavidin is perfect for tightly coupling your biotinylated protein or for capture Net Absorbance at 450 nm ™ of biotinylated oligonucleotides. MagnaBind 0 Biotin Beads are available for purification of any 0.00 0.05 0.10 0.15 biotin-binding molecule. Magnetic separation is Units src kinase a convenient, bench-top procedure for affinity separations of your molecules. Biotinylated tyrosine kinase peptide 2 was added to Reacti-Bind™ NeutrAvidin™ Coated Plates and incubated for Pkg. 30 minutes. Wells were washed; samples containing p60c-src Product # Description Size tyrosine kinase were added to phosphorylate the tyrosine ™ 21344 MagnaBind Streptavidin 5 ml residue on the peptide. Anti-phosphotyrosine monoclonal Support: 1-4 µm, iron oxide particles antibody conjugated to horseradish peroxidase was added. Capacity: 2 µg biotin/ml beads ™ ™ Tyrosine kinase activity was detected by 1-Step Turbo 21358 MagnaBind Magnet 1 TMB Substrate. Kinase activity was quantitated by compar- 96-Well Plate Separator ™ ison with a standard curve generated using the phosphory- 21359 MagnaBind Magnet 1 lated form of the same peptide substrate. for Microcentrifuge Tubes 21357 MagnaBind™ Magnet 1 for 1.5 ml Microcentrifuge Tubes References: 8, 27

Coated plates are available in bulk quantities for manufacturing applications 21 COATED PLATES (CONT.)

Reacti-Bind™ NeutrAvidin™ Reacti-Bind™ NeutrAvidin™ Coated Plate Characteristics HBC Coated Plates 96-Well Plate 384-Well Plate Unique technology for improved assay precision Binding Capacity 15 pmoles/well 10 pmoles/well Coat Volume 100 µl/well 50 µl/well Pierce’s new patent-pending plate-coating tech- Blocking Volume 200 µl/well 100 µl/well nology* offers a NeutrAvidin™ HBC Plate with a wider detection limit than our regular binding capacity plates. The standard curve exhibits Pkg. greater linearity for detecting small biotinylated Product # Description Size molecules such as peptides (see Figure) and 15129 Clear 96-Well Plates with 5 plates ® oligonucleotides resulting in greater assay preci- SuperBlock Blocking Buffer ™ 15123 Clear 96-Well Plates with 5 plates sion. Switch your assay over to Reacti-Bind Blocker™ BSA NeutrAvidin™ HBC Coated Plates for binding 15128 Clear 8-Well Strip Plates 5 plates small biotinylated ligands and see the difference ™ with Blocker BSA for yourself. 15127 Clear 8-Well Strip Plates with 5 plates SuperBlock® Blocking Buffer 15116 White 96-Well Plates 5 plates ® Notes: with SuperBlock Blocking Buffer ■ 15117 Black 96-Well Plates with 5 plates Unique plate coating technology – results SuperBlock® Blocking Buffer in high loading of NeutrAvidin™ Biotin-Binding 15400 Clear 384-Well Plates with 5 plates Protein/well ® SuperBlock Blocking Buffer ■ Improved sensitivity – less nonspecific 15401 White 384-Well Plates with 5 plates SuperBlock® Blocking Buffer binding for improved signal:noise ratios 15402 Black 384-Well Plates with 5 plates ■ Broader dynamic range – extends the quanti- ® SuperBlock Blocking Buffer tative range so there is no need for dilutions ™ 15115 Reacti-Bind Biotin Binding 4 plates ■ Plate Sample Pack Save time – pre-blocked plates to reduce One each of the following plates: the number of assay steps Product #’s 15120, 15121, 15127 and 15128 ■ Flexible assay formats – coated plates offered in 96- and 384-well formats and in different colors

Phosphopeptide Detection Assay Comparison of Biotin-Binding Protein Coated Plates 10

6 S/N

Biotinylated Phosphopeptide (pM/well)

Comparison of NeutrAvidin™ High Binding Capacity (HBC) Coated Plate, NeutrAvidin™ Regular Binding Capacity (RBC) Coated Plates and a competitor’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:noise (S/N) ratio.

22 Reacti-Bind™ NeutrAvidin™ HBC Coated Reacti-Bind™ Streptavidin Plate Characteristics Coated Polystyrene Plates 96-Well Plate 384-Well Plate The specific binding affinity of streptavidin Binding Capacity 60 pmoles/well 35 pmoles/well Coat Volume 100 µl/well 50 µl/well for biotin – in a microplate Blocking Volume 200 µl/well 100 µl/well Notes: ■ Easy and gentle immobilization of biotin conjugates Pkg. Product # Description Size ■ Low nonspecific binding 15507 Clear 96-Well Plates with 5 plates ■ No denaturing of the protein conjugate ® SuperBlock Blocking Buffer on binding 15508 Clear 8-Well Strip Plates with 5 plates ■ ™ SuperBlock® Blocking Buffer Preblocked with your choice of Blocker 15509 White 96-Well Plates with 5 plates BSA or SuperBlock® Blocking Buffer SuperBlock® Blocking Buffer 15510 Black 96-Well Plates with 5 plates ® Reacti-Bind™ Streptavidin Coated SuperBlock Blocking Buffer 15511 Clear 384-Well Plates with 5 plates Plate Characteristics ® SuperBlock Blocking Buffer 96-Well Plate 384-Well Plate 15512 White 384-Well Plates with 5 plates Binding Capacity 5 pmoles/well 4 pmoles/well SuperBlock® Blocking Buffer Coat Volume 100 µl/well 50 µl/well 15513 Black 384-Well Plates with 5 plates SuperBlock® Blocking Buffer Blocking Volume 200 µl/well 100 µl/well *U.S. patent pending on high-binding capacity plate-coating technology. Pkg. Product # Description Size For immediate assistance, visit www.piercenet.com 15120 Clear 8-Well Strip Plates with 5 plates or call 800-874-3723. Outside the United States, contact SuperBlock® Blocking Buffer your local Perbio Science branch office or distributor. 15121 Clear 8-Well Strip Plates 5 plates with Blocker™ BSA 15122 Clear 8-Well Strip Plates with 25 plates SuperBlock® Blocking Buffer 15124 Clear 96-Well Plates with 5 plates SuperBlock® Blocking Buffer 15125 Clear 96-Well Plates with 5 plates Blocker™ BSA 15126 Clear 96-Well Plates with 25 plates SuperBlock® Blocking Buffer 15118 White 96-Well Plates with 5 plates SuperBlock® Blocking Buffer 15119 Black 96-Well Plates with 5 plates SuperBlock® Blocking Buffer 15405 Clear 384-Well Plates with 5 plates SuperBlock® Blocking Buffer 15406 White 384-Well Plates with 5 plates SuperBlock® Blocking Buffer 15407 Black 384-Well Plates with 5 plates SuperBlock® Blocking Buffer 15115 Reacti-Bind™ Biotin 4 plates Binding Plate Sample Pack One each of the following plates: Product #’s 15120, 15121, 15127 and 15128 Reference: 17

Coated plates are available in bulk quantities for manufacturing applications 23 COATED PLATES (CONT.)

Reacti-Bind™ Streptavidin Notes: HBC Coated Plates ■ Broader dynamic range – extends the quanti- tative range so there is no need for dilutions Take advantage of a new Pierce technology that ■ Better sensitivity – increased binding capacity provides a broader dynamic range allows direct detection of small ligands not Reacti-Bind™ Streptavidin High Binding Capacity observed with regular binding capacity plates (HBC) Coated Plates are designed for binding ■ Superior assay precision – standard curve biotinylated oligonucleotides and peptides with demonstrates greater linearity higher binding efficiency than other commercial- ■ Save time – pre-blocked plates to reduce ly available plates. Pierce’s coating technology the number of assay steps (patent pending)* has created a streptavidin- ■ Flexible assay formats – coated plates coated plate with four- to five-times the binding offered in 96- and 384-well formats and capacity of competitors’ plates. Using a Reacti- in different colors Bind™ Streptavidin HBC Plate can result in an assay with a broader dynamic range and better Reacti-Bind™ Streptavidin HBC Coated linearity, leading to improved assay precision. Plate Characteristics 96-Well Plate 384-Well Plate Binding Capacity 125 pmoles/well 60 pmoles/well Comparison of Streptavidin High Binding Coat Volume 100 µl/well 50 µl/well Capacity (HBC) and Competitor’s HBC Coated Plates Blocking Volume 200 µl/well 100 µl/well 180 160 140 Pkg. Product # Description Size 120 15500 Clear 96-Well Plates with 5 plates 100 SuperBlock® Blocking Buffer S/N 80 15501 Clear 8-Well Strip Plates with 5 plates ® 60 SuperBlock Blocking Buffer 15502 White 96-Well Plates with 5 plates 40 SuperBlock® Blocking Buffer 20 15503 Black 96-Well Plates with 5 plates ® 0 SuperBlock Blocking Buffer 15504 Clear 384-Well Plates with 5 plates SuperBlock® Blocking Buffer Fluoresceinated Oligonucleotide (pM/well) 15505 White 384-Well Plates with 5 plates SuperBlock® Blocking Buffer Fluoresceinated oligonucleotide hybridization assay com- 15506 Black 384-Well Plates with 5 plates parison. Comparison of Reacti-Bind™ Streptavidin High SuperBlock® Blocking Buffer Binding Capacity (HBC) Coated Plate with competing *U.S. patent pending on high-binding capacity plate-coating tech- High Binding Capacity plate (CHC). Plates were incubat- nology. ed 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:noise (S/N) ratio.

Coated plates are available in bulk quantities for manufacturing applications 24 Reacti-Bind™ Biotin Coated NeutrAvidin™, Horseradish Microplates Peroxidase Conjugated Notes: Notes: ■ Biotin group accessible for binding ■ Lower nonspecific binding than avidin, streptavidin or NeutrAvidin™ streptavidin conjugates Biotin-Binding Protein ■ Better signal-to-noise ratio in assay systems ■ Pre-blocked to reduce nonspecific binding ■ 1-2 moles HRP/mole conjugate ■ Strip well plate format for the ultimate ■ 3-8 µg biotin bound/mg conjugate in convenience ■ ≥80 peroxidase units/mg conjugate ■ Coat volume: 200 µl Pkg. ■ Blocking volume: 300 µl Product # Description Size 31001 NeutrAvidin™, Horseradish 2 mg Peroxidase Conjugated Pkg. References: 11, 18, 25, 69 Product # Description Size 15151 Clear 8-Well Strip Plates 5 plates

NeutrAvidin™, Alkaline Phosphatase Conjugated ™ NEUTRAVIDIN CONJUGATES Notes: ■ Lower nonspecific binding than NeutrAvidin™, Streptavidin and streptavidin conjugates Avidin Conjugates ■ Better signal-to-noise ratio in assay systems ■ 1-2 moles HRP/mole conjugate Avidin conjugates offer several advantages when ■ 3-8 µg biotin bound/mg conjugate used in a variety of detection systems. The ■ ≥100 phosphatase units/mg conjugate incredible specificity of avidin-biotin binding, combined with its high affinity, allow this interac- Pkg. Product # Description Size tion to be harnessed in place of antibodies to pro- 31002 NeutrAvidin™, Alkaline 2 mg duce lower backgrounds and higher Phosphatase Conjugated signal-to-noise ratios in assays such as References: 11, 18, 25, 69 Western/Southern/Northern blotting, ELISA, immunohistochemical staining, and fluorescence activated cell sorting. NeutrAvidin™, FITC Conjugated

ImmunoPure® NeutrAvidin™ Notes: Biotin-Binding Protein ■ Fluorescent-labeled NeutrAvidin™ Protein ■ ≥ 2 moles FITC/mole NeutrAvidin™ Protein Notes: ■ Absorption: 490 nm ■ pl has been reduced to a neutral state ■ Emission: 520 nm ■ Deglycosylated, so lectin binding is reduced ■ Color: greenish-yellow to undetectable levels Pkg. ■ Can be used as a biotin blocking agent in Product # Description Size tissues for histochemistry 31006 NeutrAvidin™, 5 mg ■ 11-17 µg biotin bound/mg NeutrAvidin™ FITC Conjugated Biotin-Binding Protein

Pkg. For immediate assistance, visit www.piercenet.com Product # Description Size or call 800-874-3723. Outside the United States, contact ® ™ 31000 ImmunoPure NeutrAvidin 10 mg your local Perbio Science branch office or distributor. Biotin-Binding Protein References: 21, 22 25 ™ NEUTRAVIDIN CONJUGATES (CONT.)

EZ-Link® Maleimide ImmunoPure® Avidin, Horseradish Activated NeutrAvidin™ Peroxidase Conjugated Biotin-Binding Protein Notes: Notes: ■ Purified using special affinity techniques ■ Great for preparing NeutrAvidin™ to eliminate nucleic acids conjugates of peptides or proteins ■ 1-2 moles HRP/mole avidin with free sulfhydryl (–SH) groups ■ 5-10 µg biotin bound/mg of protein ■ Reacts with sulfhydryls in the range ■ ≥80 peroxidase units/mg conjugate of pH 6.5-7.5 Pkg. ■ Extremely low nonspecific binding Product # Description Size characteristics 21123 ImmunoPure® Avidin, 2 mg ■ ™ Horseradish Peroxidase 4-8 moles maleimide/mole NeutrAvidin Protein Conjugated ■ ™ 18-22 µg biotin bound/mg NeutrAvidin Protein 29994 ImmunoPure® Avidin, 5 mg Horseradish Peroxidase Pkg. Conjugated Product # Description Size 31007 EZ-Link® Maleimide 5 mg Activated NeutrAvidin™ Protein ImmunoPure® Avidin, Alkaline Phosphatase Conjugated Notes: ■ Homogeneous by SDS-PAGE ■ Purified using special affinity techniques AVIDIN CONJUGATES to eliminate nucleic acids ■ ~/mole alkaline phosphatase/mole avidin ® ImmunoPure Avidin ■ One unit = 1.0 micromole of p-nitrophenol Notes: liberated from p-nitrophenylphosphate per minute at 37°C, pH 9.5 ■ Hen egg white glycoprotein, affinity-purified, salt-free, lyophilized powder Pkg. Product # Description Size ■ Molecular mass of 67,000-68,000 daltons 21321 ImmunoPure® Avidin, 100 ■ Isoelectric point of 10-10.5 Alkaline Phosphatase units ■ Stable over a wide range of pH and temperature Conjugated ■ Blocking protein for biotin-rich tissue sections

(use at 0.1% for inhibition of endogenous biotin) ® ■ 11-14 µg biotin bound/mg avidin ImmunoPure Avidin, Fluorescein (FITC) Conjugated Pkg. Product # Description Size Notes: ■ 21121 ImmunoPure® Avidin 10 mg Fluorescent-labeled avidin 21128 ImmunoPure® Avidin 20 mg ■ ~3.5 moles fluorescein/mole avidin Reference: 70 ■ Absorption: 490 nm ■ Emission: 520 nm ■ Color: greenish-yellow ■ No free fluorescein Pkg. Product # Description Size 21221 ImmunoPure® Avidin, 5 mg Fluorescein (FITC) Conjugated

26 ImmunoPure® Avidin, Streptavidin, Alkaline R-Phycoerythrin Conjugated Phosphatase Conjugated Notes: Notes: ■ Fluorescent-labeled avidin ■ ≥3 µg biotin bound/mg conjugate ■ Absorption: 450-570 nm ■ ≥100 phosphatase units/mg conjugate ■ Emission: 574 nm Pkg. ■ Color: Red Product # Description Size 21324 Streptavidin, Alkaline 1 mg Pkg. Phosphatase Conjugated Product # Description Size 21323 Streptavidin, Alkaline 3 mg 21021 ImmunoPure® Avidin, 1 mg Phosphatase Conjugated R-Phycoerythrin Conjugated Reference: 70

Streptavidin, STREPTAVIDIN CONJUGATES Fluorescein (FITC) Conjugated Notes: ® ImmunoPure Streptavidin ■ Fluorescent-labeled streptavidin Notes: ■ 3-5 moles FITC/mole streptavidin ■ Much less soluble in water than avidin ■ Excitation: 490 nm ■ 13-22 µg biotin bound/mg of protein ■ Emission: 520 nm ■ Recombinant ■ Color: greenish-yellow

Pkg. Pkg. Product # Description Size Product # Description Size 21224 Streptavidin, 1 mg 21122 ImmunoPure® Streptavidin 1 mg ® Fluorescein (FITC) 21125 ImmunoPure Streptavidin 5 mg Conjugated

Streptavidin, Horseradish Peroxidase Conjugated Streptavidin, Rhodamine Notes: Conjugated (TRITC) ■ 1-2 moles HRP/mole of streptavidin Notes: ■ ≥ 100 peroxidase units/mg of conjugate ■ Fluorescent-labeled streptavidin ■ 6-9 µg biotin bound/mg conjugate ■ 1-3 moles TRITC/mole streptavidin ■ Absorption: 515-520 nm and 550-555 nm Pkg. Product # Description Size ■ Emission: 575 nm 21126 Streptavidin, Horseradish 1 mg ■ Color: orange-red Peroxidase Conjugated 21124 Streptavidin, Horseradish 2 mg Pkg. Peroxidase Conjugated Product # Description Size 21127 Streptavidin, Horseradish 5 mg 21724 Streptavidin, 1 mg Peroxidase Conjugated Rhodamine Conjugated (TRITC) Reference: 12

For immediate assistance, visit www.piercenet.com or call 800-874-3723. Outside the United States, contact your local Perbio Science branch office or distributor.

Coated plates are available in bulk quantities for manufacturing applications 27 STREPTAVIDIN CONJUGATES (CONT.)

® Streptavidin, detect this antibody should be the ImmunoPure ® ABC Mouse IgG Kit. To create a sensitive detec- Texas Red Conjugated tion system, you need your specific primary anti- Notes: body, an ImmunoPure® ABC Mouse Kit with ■ Fluorescent-labeled streptavidin biotinylated antibody, and an enzyme substrate. ■ Absorption: 595 nm The standard kit includes only the avidin and ■ Emission: 615 nm biotinylated enzyme and is useful if an ABC Kit is ■ Color: red not available for your specific species of primary Pkg. antibody, or if the primary antibody is already Product # Description Size labeled with biotin. The biotinylated antibody can 21624 Streptavidin, 1 mg then be used, along with a blocking agent, and Texas Red® Conjugated used with a standard kit. Texas Red® is a registered trademark of Molecular Probes.

The ImmunoPure® ABC Kits contain: ImmunoPure® ■ 3 ml of the appropriate blocking serum Streptavidin Hydrazide ■ 1 ml of the biotinylated affinity-purified secondary antibody Notes: ■ 2 ml each of the avidin and biotinylated ■ Covalently attaches streptavidin to oxidized enzyme reagents carbohydrate moieties on glycoproteins ■ ≥4 moles hydrazide/mole streptavidin The Ultra-Sensitive ABC Peroxidase Staining Kits are more sensitive than the ABC Peroxidase Staining Kits, without exhibiting increased back- Pkg. Product # Description Size ground staining. These kits supply the extra sen- 21120 ImmunoPure® 2 mg sitivity needed for localizing antigens present in Streptavidin Hydrazide 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.

ABC STAINING KITS Comparison of the Relative Sensitivities of Three Amplification Methods ® ImmunoPure ABC Staining Kits are highly sensi- vs. a Direct ELISA Technique tive, have rapid avidin-biotin interactions and produce very low background staining. Highly 2.00 diluted primary antibodies can be used with 1.75 Direct ELISA LAB ELISA Pierce ImmunoPure® Staining Kits, producing 1.50 BRAB ELISA comparable stain intensity to other methods that 1.25 ABC ELISA require higher concentrations of antibody. 1.00 Two types of enzymatic ABC staining kits, 0.75 0.50 alkaline phosphatase and horseradish peroxi- Absorbance at 405 nm dase, are available. The kits can come with 0.25 or without (standard kit) a biotinylated 0.00 secondary antibody. 0 250 500 750 1,000 1,250 1,500 Antigen (ng) 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 Coated plates are available in bulk quantities for manufacturing applications 28 BIOTIN CONJUGATES

Pkg. Product # Description Size Biotin Fluorescein 32028 ImmunoPure® ABC Kit Peroxidase Mouse IgG Staining Kit Includes: Biotinylated Anti-Mouse 31.62 Å IgG Antibody Blocking Buffer O Avidin HN Biotinylated HRP NH H 32052 ImmunoPure® Kit H H O HO Ultra-Sensitive ABC N O S O N O Peroxidase Mouse H O IgG Staining Kit O Includes: Biotinylated Anti-Mouse IgG Antibody Blocking Buffer O Avidin (+)-Biotinyl-fluorescein 8.83 Å Biotinylated HRP M.W. 732.80 HO 32032 ImmunoPure® ABC Kit Peroxidase Rabbit IgG Staining Kit Notes: Includes: Biotinylated Anti-Rabbit IgG Antibody Blocking Buffer ■ Water-soluble Avidin ■ Perfect for diagnostic applications Biotinylated HRP ■ 32054 ImmunoPure® Kit Absorbance: 492 nm Ultra-Sensitive ABC ■ Color: greenish-yellow Peroxidase Rabbit IgG Staining Kit Pkg. Includes: Biotinylated Anti-Rabbit Product # Description Size IgG Antibody Blocking Buffer 22030 Biotin Fluorescein 5 mg Avidin Biotinylated HRP ® 32020 ImmunoPure ABC Kit Continued on next page Standard Peroxidase Staining Kit Includes: Avidin Biotinylated HRP 32050 ImmunoPure® Kit Ultra-Sensitive ABC Standard Peroxidase Staining Kit Includes: Avidin Biotinylated HRP 32044 ImmunoPure® ABC Kit Alkaline Phosphatase Mouse IgG Staining Kit Includes: Biotinylated Anti-Mouse IgG Antibody Blocking Buffer Avidin Biotinylated Alkaline Phosphatase 32046 ImmunoPure® ABC Kit Alkaline Phosphatase Rabbit IgG Staining Kit Includes: Biotinylated Anti-Rabbit IgG Antibody Blocking Buffer Avidin Biotinylated Alkaline Phosphatase 32040 ImmunoPure® ABC Kit Standard Alkaline Phosphatase Staining Kit Includes: Avidin Biotinylated Alkaline Phosphatase

For immediate assistance, visit www.piercenet.com or call 800-874-3723. Outside the United States, contact your local Perbio Science branch office or distributor.

29 BIOTIN CONJUGATES (CONT.)

Pkg. Product # Description Notes Size 29130 ImmunoPure® ■ Commonly used as a molecular weight marker on 25 mg Biotinylated SDS-PAGE and in gel permeation Bovine Serum ■ Frequently used as a blocking agent in many immunological techniques Albumin (BSA) ■ 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 29139 ImmunoPure® ■ An extremely pure reagent with virtually all enzyme 5 mg Biotinylated activity retained after biotinylation Horseradish ■ Offers excellent sensitivity and is recommended for use in sandwich techniques Peroxidase that utilize ImmunoPure® 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 ImmunoPure® ■ Alkaline phosphatase has been isolated from calf intestine 1 mg Biotinylated ■ Biotinylation of this enzyme does not alter its high specific activity Alkaline ■ Can be used as a secondary marker in immunohistochemical staining Phosphatase ■ 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 29939 ImmunoPure® ■ Used with ONPG for histochemical and 100 units Biotinylated immunoblotting applications β-Galactosidase ■ 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 29988 ImmunoPure® ■ Useful as a substitute for biotinylated secondary antibodies 0.5 mg Biotinylated ■ Can detect or locate immunoglobulins on cell surface or in tissue Protein G ■ Recombinant form of Protein G, altered to remove the albumin binding sites to reduce nonspecific binding 29989 ImmunoPure® ■ Useful as a substitute for biotinylated secondary antibodies 1 mg Biotinylated ■ Can detect or locate immunoglobulins on cell surface or in Protein A tissue cell surface or in tissue

® 29997 ImmunoPure ■ Binds to the VL region of kappa light chains (human I, III, IV .5 mg Biotinylated and Mouse I) without interfering with antigen-binding sites Protein L ■ 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) 31826 ImmunoPure® ■ Prepared with highly purified IgG 5 mg Biotinylated ■ Detect low concentrations of Fc receptors or Rabbit IgG 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 29129 ImmunoPure® ■ Free biotin is used in the elution buffer to purify biotinylated 1 g D-Biotin compounds from monomeric avidin columns ■ Can be used as a reference standard for the HABA dye assay when measuring degree of biotinylation

ImmunoPure® Biotinylated Secondary Antibodies Pierce offers 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.piercenet.com

For immediate assistance, visit www.piercenet.com or call 800-874-3723. Outside the United States, contact your local Perbio Science branch office or distributor.

30 EXAMPLE PROTOCOLS FOR BIOTINYLATION

Additional protocols can be found in individual Materials required: product instruction booklets that can be down- ■ Biotinylation reagent such as Sulfo-NHS-LC- loaded from www.piercenet.com. Biotin (Product # 21335) The molar ratio of biotinylation reagent to protein ■ PBS (0.1 M phosphate, 0.15 M NaCl; pH 7.2; is one of the most important parameters to con- or Product # 28372) or other non-amine consider when biotin-labeling a protein. This ratio taining buffer at pH 7.0-8.5 (recommend pH will determine the degree of labeling that is 7.5-8.0) achieved. A high degree of labeling can ensure ■ Device for removal of unreacted biotin such that each protein molecule contains at least one as Slide-A-Lyzer® Dialysis Cassette Kits* for biotin moiety. However, a low degree of biotiny- dialysis of 0.5-3.0 ml samples (Product # lation ensures minimal modification of the native 66382) or D-Salt™ Dextran Desalting protein, resulting in maximum retention of bio- Columns, 5K MWCO, for desalting of sam- logical activity. For large proteins such as anti- ples up to 1.25 ml (Product # 43230) bodies, a modification level of three to five Procedure: biotins/protein is generally optimal, while small proteins may require slightly less modification. 1.Allow the biotinylation reagent to warm The optimal biotinylation level for any protein completely to room temperature before can only be determined experimentally. opening the vial. 2.Dissolve 2-10 mg of IgG in 1 ml PBS.

® 3.Immediately before use, make a 10 mM Biotinylating IgG with EZ-Link stock solution of Sulfo-NHS-LC-Biotin. Sulfo-NHS-Biotin Reagents 4.Add sufficient volume of the concentrated Preparing stock solutions of any NHS-esters of Sulfo-NHS-LC-Biotin to give a 12-fold molar biotin with the intent of long-term storage is not excess of biotin to a 10 mg/ml IgG solution or a 20-fold molar excess of biotin to a recommended because hydrolysis occurs very 2 mg/ml IgG solution (See Table 1 for quickly in solution. The container of biotinylation examples). reagent must be brought to room temperature prior to opening, then the proper amount of 5.Place the reaction mixture on ice and incu- biotinylation reagent is weighed, dissolved and bate for two hours. Alternatively, incubate at used immediately. Even those NHS-esters that room temperature for 30 minutes. require organic solvents should be made up 6.Use dialysis or gel filtration to remove the immediately before use as these solvents (i.e., unreacted Sulfo-NHS-LC-Biotin. See instruc- DMSO and DMF) are hygroscopic and water tions provided with preferred buffer absorbed from the air results in hydrolysis of the exchange product. reagent. 7.Store the biotinylated protein under the same condition that is optimal for the non- These conditions generally result in an biotinylated protein. immunoglobulin with a modification level of *Slide-A-Lyzer® Dialysis Cassette Technology is approximately three to five biotins per molecule protected by U.S. Patent # 5,503,741. of IgG. The molar ratio of Sulfo-NHS-Biotin to protein may be adjusted to achieve a particular outcome.

31 EXAMPLE PROTOCOLS FOR BIOTINYLATION (CONT.)

Table 1. Amount of biotinylation reagent to add to a 1 ml protein sample. Protein M.W. of mg mMole Fold Molar mM Biotin Volume of Protein Protein/ml Protein/ml Excess of Reagent 10 mM Biotin Biotin to Add Reagent to Add Protein A 42,000 10 2.4 x 10-4 12 2.9 x 10-3 290 µl 24.8 x 10-5 20 9.5 x 10-4 95 µl BSA 68,000 10 1.5 x 10-4 12 1.8 x 10-3 180 µl 23.0 x 10-5 20 5.9 x 10-4 59 µl IgG 150,000 10 6.7 x 10-5 12 8.0 x 10-4 80 µl 21.3 x 10-5 20 2.7 x 10-4 27 µl

Table 2. Troubleshooting guide for biotinylation with NHS-esters. Problem Cause Solution Poor biotinylation No amines available on ■ Choose biotinylation reagent that targets molecule of interest different group. ■ Convert sulfhydryl to amine using Aminoethyl-8 (Product # 23010). Inappropriate choice ■ Choose non-amine-containing buffer at pH 7-9 of buffer (hydrolysis is very rapid at higher pH). Hydrolysis of ■ Allow reagent to come to RT before opening. biotinylation reagent ■ Make up fresh biotinylation reagent. ■ Use biotinylation reagent immediately. Incomplete removal of ■ Dialyze or desalt thoroughly into primary amines non-amine-containing buffer at pH 7-9. Protein loses Over-biotinylation ■ Reduce molar excess of biotinylation reagent. function ■ Reduce time or temperature for biotinylation. ■ Choose biotinylation reagent that targets different groups.

32 Biotinylating Cell Surface Proteins Procedure: Selective labeling of proteins located at the cell 1. Wash cells three times with ice-cold PBS, surface is often desirable in the study of integral pH 8.0, to remove any contaminating proteins. membrane proteins. Cell surface biotinylation 2.Suspend the cells at a concentration of has emerged as an important tool for studying ~25 x 106 cells/ml in PBS, pH 8.0. the expression and regulation of receptors and Note: Other cell concentrations can be used. transporters, differentiation of plasma mem- The concentration of biotinylation reagent can brane proteins from those localized to organelle be scaled up or down accordingly, based on membranes, and distribution of membrane pro- cell size, type, etc. teins in polarized epithelial cells. The specificity of Sulfo-NHS-esters of biotin for cell surface 3.Add 1 mg of Sulfo-NHS-LC-Biotin per ml of labeling has been demonstrated in these applica- reaction volume. tions.13, 26 A variety of similar protocols have 4.Incubate at room temperature for 30 minutes. been used successfully on a wide range of cell Note: Performing this incubation at 4°C may types.2, 13, 26, 37, 40, 52, 54 Because these molecules reduce active internalization of the biotinyl- dissolve readily in polar solutions and are ation reagent. charged by the sodium sulfoxide group on the succinimidyl ring, they cannot permeate the cell 5.Wash cells three times with ice-cold PBS + membrane. As long as the cell is intact, only pri- 100 mM glycine to quench any remaining mary amines exposed on the surface are biotiny- biotinylation reagent. lated. For experiments in which internal The cell surface proteins are now biotinylated ® biotinylation is desired, EZ-Link NHS-LC-Biotin on exposed lysine residues. (Product # 21336), or other non-water-soluble analogs, can be used.

Following cell surface biotinylation, it is often Pierce offers a Cell Surface Protein Biotinylation necessary to isolate the biotinylated proteins. and Purification Kit, which is a convenient way This is best accomplished using immobilized to biotin-label and purify mammalian cell-sur- NeutrAvidin™ Biotin-Binding Protein, when face proteins. This kit efficiently labels proteins harsh elution conditions are appropriate, or with accessible lysine residues and sufficient Monomeric Avidin, when a milder elution is indi- extracellular exposure. The kit includes all the cated for the recovery of functional protein mol- reagents necessary for labeling, cell lysis and ecules. Several example protocols for affinity purification of cell-surface proteins. purification of biotinylated molecules are given in the following pages of this booklet. Pkg. Product # Description Size Materials required: 89881 Cell Surface Protein Kit Biotinylation and ■ Sulfo-NHS-LC-Biotin (Product # 21335) or Purification Kit other Sulfo-NHS-ester biotinylation reagent Includes: EZ-Link® Sulfo-NHS-SS-Biotin 8 x 12 ■ PBS (0.1 M phosphate, 0.15 M NaCl; pH 7.2; mg vials Quenching Solution 16 ml or Product # 28372) Lysis Buffer 4.5 ml Immobilized NeutrAvidin™ Gel 2.25 ml gel slurry Wash Buffer 34 ml Column Accessory Pack 8 spin columns No-Weigh™ Dithiothreitol (DTT) 8 x 7.7 mg BupH™ Phosphate Buffered Saline 2 packs BupH™ Tris Buffered Saline 1 pack

33 EXAMPLE PROTOCOLS FOR BIOTINYLATION (CONT.)

One-Step Biotinylation and Procedure: Dialysis in a Slide-A-Lyzer® 1. Allow the biotinylation reagent to warm Cassette completely to room temperature before opening the vial. Small-volume samples have the inherent problem of sample loss due to handling. This can be 2. Immediately before use, make a 10 mM an especially important complication if several solution of Sulfo-NHS-LC-Biotin. processing procedures are required. The use of 3. Add biotinylation reagent directly to the a Slide-A-Lyzer® Dialysis Cassette (SAL) can protein in PBS. Add sufficient volume of the prevent sample loss because more than one 10 mM Sulfo-NHS-LC-Biotin to give a 12-fold reaction can be performed in a single container, molar excess of biotin to a 10 mg/ml IgG thus eliminating transfer steps. The following is solution or a 20-fold molar excess of biotin a sample protocol for biotinylating a small to a 2 mg/ml IgG solution (See Table 1 for amount of protein. A similar method can be examples). used for other reactions inside the SAL. Note: Buffers other than PBS can be used, Materials required: provided they are at pH 7.0-8.5 and do not contain primary amines. ■ 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) 5. Tape the SAL onto a shaking platform and incubate for 30 minutes to 1 hour at room ■ SAL Kit (Product # 66382, includes cas- temperature or 2 hours at 4°C, shaking so settes, syringes, needles and buoys) that the bubble can be seen “mixing” the ■ Protein solution (This protocol is written for reagents. 1 ml of a 2-10 mg/ml solution. The choice of 6. Remove SAL from the shaking platform, SAL and amount of biotinylation reagent can insert the needle into another port and with- be adjusted to suit other protein amounts.) draw 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.

34 Biotinylation Troubleshooting Guide Problem Possible Cause Solution No Avidin binding Insufficient biotinylation ■ 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 ■ Use long chain (LC or LC-LC) analog of too short biotinylation reagent. Loss of 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 from amine-reactive to sulfhydryl-reactive reagent).

35 EXAMPLE PROTOCOLS FOR AFFINITY PURIFICATION BASED ON AVIDIN-BIOTIN BINDING Introduction Affinity Purification of Biotinylated Affinity purification makes use of a specific bind- Molecules (Column Format) ing interaction that occurs between molecules Materials required: and that is used extensively for the isolation of ■ biological molecules. A single pass through an Immobilized Avidin, Streptavidin or affinity column can achieve a 1,000- to 10,000- NeutrAvidin™ Biotin-Binding Protein fold purification of ligand from a crude mixture. ■ Biotinylated sample in solution (~3 mg From a single affinity purification step, it is pos- biotinylated protein/ml immobilized biotin- sible to isolate a compound in a form pure binding protein). enough to obtain a single band upon SDS-PAGE ■ Binding buffer: Phosphate Buffered Saline analysis. (0.1 M phosphate, 0.15 M NaCl; pH 7.2; Product # 28372). To reduce nonspecific In affinity purification, a ligand is immobilized to binding, add 1% of a detergent such as a solid support. Once immobilized, it specifically NP-40 to the binding buffer. binds its partner under mild buffer conditions ■ Elution buffer: 8 M guanidine•HCl, pH 1.5 (often physiologic conditions such as phosphate (Product # 24115) buffered saline). After binding to the partner ■ molecule, the support is washed with additional Columns: Disposable Polystyrene Columns (Product # 29920 for gel volumes of 2 ml or buffer to remove unbound components of the less or Product # 29924 for gel volumes of sample. An elution buffer is added. The elution 2-10 ml) buffer disrupts the interaction between the lig- and and its binding partner by pH extremes (low or high), high salt, presence of detergents, Procedure: chaotropic agents that unfold one or both of the 1. Pack gel into the column. molecules, or the removal of some factor 2. Equilibrate the column with three column required for the pair to bind. Once released, the volumes of binding buffer. binding partner can be recovered from the sup- 3. Add biotinylated sample to the column and port using additional elution buffer. The buffer allow sample to enter the gel bed. can then be exchanged by dialysis or desalting Sequentially replace the bottom and top into a more suitable buffer for storage or down- caps and incubate at room temperature for stream analysis. 30 minutes. Any molecule that has an interacting partner can Note: If the sample is large enough that the be attached to a support and used for affinity entire amount cannot be added at once, purification. The extraordinary affinity of avidin incubate for 10-15 minutes and allow some for biotin allows biotin-containing molecules to of the solution to pass through the column. be discretely bound to immobilized avidin. Once Add more sample and incubate. Do not exceed the binding capacity of the gel. biotin is attached to a molecule, the molecule can be affinity purified using an immobilized ver- 4. Wash the column with 10 column volumes of sion of any biotin-binding protein. Alternatively, binding buffer. a biotinylated molecule can be immobilized 5. Elute the biotinylated molecules with 5-10 through interaction with a biotin-binding protein, column volumes of the elution buffer. Collect then used to affinity purify other molecules that the eluate in 0.5-1 ml fractions. Monitor specifically interact with it. 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.

36 Affinity Purification Using a Procedure: Biotinylated Antibody 1. Pack gel into the column. (Column Format) 2. Equilibrate the column with three column volumes of binding buffer. Materials required: 3. Add biotinylated antibody to the column and ■ Immobilized Avidin, Streptavidin or allow solution to enter the gel bed. Replace NeutrAvidin™ Biotin-Binding Protein the bottom and top caps sequentially and ■ Biotinylated antibody in solution (~3 mg incubate at room temperature for 30 minutes. biotinylated antibody/ml immobilized biotin- Note: If the sample is large enough that the binding protein). entire amount cannot be added at once, ■ Binding buffer: Phosphate Buffered Saline incubate for 10-15 minutes and allow some (0.1 M phosphate, 0.15 M NaCl; pH 7.2; of the solution to pass through the column. Product # 28372). To reduce nonspecific Add more antibody and incubate. Do not binding, add 1% of a detergent such as exceed the binding capacity of the gel. NP-40. 4. Wash the column with 10 column volumes of ■ Elution buffer: ImmunoPure® IgG Elution binding buffer. ® Buffer (Product # 21004), ImmunoPure 5. Add antigen-containing sample to the column Gentle Ag/Ab Elution Buffer (Product # and allow it to enter the gel bed. Replace the 21027) or 0.1 M glycine•HCl, pH 2.8 bottom and top caps sequentially and incu- ■ Columns: Disposable Polystyrene Columns bate at room temperature for 1-2 hours. (Product # 29920 for gel volumes of 2 ml or 6. Wash the column with 10 column volumes of less or Product # 29924 for gel volumes of binding buffer. 2-10 ml) 7. Elute the antigen with 5-10 column volumes elution buffer. Collect the eluate in 0.5-1 ml fractions. If using ImmunoPure® 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. Note: If using ImmunoPure® 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.

37 EXAMPLE PROTOCOLS FOR AFFINITY PURIFICATION (CONT.) Immunoprecipitation Using a Procedure: Biotinylated Antibody 1. In a microcentrifuge tube, solubilize antigen in 50 µl of binding buffer and add the biotiny- (Batch Format) lated antibody. Adjust the volume of the Materials required: sample to 0.2 ml with binding buffer. ■ Immobilized Avidin, Streptavidin or 2. Incubate the sample overnight at 4°C. NeutrAvidin™ Biotin-Binding Protein 3. Mix the immobilized avidin to ensure an even ■ Biotinylated sample in solution (~3 mg suspension. Add the appropriate amount of biotinylated protein/ml immobilized biotin- immobilized avidin to the tube containing the binding protein). antigen/biotinylated antibody mixture. ■ Binding buffer: Phosphate Buffered Saline 4. Incubate the sample with mixing for 1 hour at (0.1 M phosphate, 0.15 M NaCl; pH 7.2; room temperature or 4°C. Product # 28372). To reduce nonspecific 5. Wash the avidin-bound complex with 0.5- binding, add 1% of a detergent such as 1.0 ml of binding buffer and centrifuge for 1- NP-40. 2 minutes at approximately 2,500 x g. ■ Elution buffer: ImmunoPure® IgG Elution Remove the supernatant. Repeat this wash Buffer (Product # 21004), ImmunoPure® procedure at least four times and remove the Gentle Ag/Ab Elution Buffer (Product # final wash. 21027) or 0.1 M glycine•HCl, pH 2.8 6. Add elution buffer to the gel to recover the ■ Microcentrifuge tubes or Handee™ Spin Cup bound antigen. If using ImmunoPure® IgG Columns (Product # 69700) 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).

38 REFERENCES

1. Alley, S.C., et al. (2000). J. Am. Chem. Soc. 122, 6126- 37. Leighton, B.H., et al. (2002). J. Biol. Chem. 277, 29847- 6127. 29855. 2. Altin, J.G., et al. (1995). Anal. Biochem. 224, 382-389 38. Lesa, G.M., et al. (2000). J. Biol. Chem. 275, 2831- 3. Araga, S., et al. (1999). J. Immunol. 163, 476-482. 2836. 4. Arosa, F.A., et al. (1999). J. Biol. Chem. 274, 16917- 39. Liaw, P.C.Y., et al. (2001). J. Biol Chem. 276, 8364- 16922. 8370. 5. Baqui, M., et al. (2003). J. Biol. Chem. 278, 1206-1211. 40. Liu, L.A. and Engvall, E. (1999). J. Biol. Chem. 274, 38171-38176. 6. Bayer, E.A. et al. (1988). Anal. Biochem. 170, 271-281. 41. Liu, Y., et al. (1999). Proc. Natl. Acad. Sci. U.S.A. 96, 7. Cernuda-Morollon, E., et al. (2001). J. Biol. Chem. 276, 14694-14699. 35530-35536. 42. Luk, J.M., et al. (1995). Anal. Biochem. 232, 217-224. 8. Chaudhuri, T.K., et al. (2001). Cell 107, 235-246. 43. Minami, Y., et al. (2000). J. Biol. Chem. 275, 9055- 9. Chiu, N.H., et al. (1999). Clin. Chem. 45, 1954-1959. 9061. 10. Cimono, G.D., et al. (1985). Annu. Rev. Biochem. 54, 44. Muroi, M., et al. (2002). J. Biol. Chem. 277, 42372- 1151-1193. 42379. 11. Claypool, S.M., et al. (2002). J. Biol. Chem. 277, 28038- 45. Neely, K.E., et al. (2002). Mol. Cell. Biol. 22, 1615-1625. 28050. 46. Nielsen, P.K., et al. (2000). J. Biol. Chem. 275, 14517- 12. Conti, L.R., et al. (2001). J. Biol. Chem. 276, 41270- 14523. 41278. 47. Nunomura, W., et al. (2000). J. Biol. Chem. 275, 6360- 13. Daniels, G.M. and Amara, S.G. (1998). Methods. 6367. Enzymol. 296, 307-318. 48. Oda, Y, et al. (2001). Nat. Biotechnol. 19, 379-382. 14. Daum, J.R., et al. (2000). Curr. Biol. 10, 850-852. 49. Orr, G.A., et al. (1981). J. Biol. Chem. 256, 761-766. 15. Edwards, S.W., et al. (1999). J. Biol. Chem. 274, 16331- 16336. 50. Reisfeld, A., et al. (1987). Biochem. Biophys. Res. Commun. 142, 519-526. 16. Ellerbroek, S.M., et al. (2001). J. Biol. Chem. 276, 24833-24842. 51. Roffman, E., et al. (1986). Biochem. Biophys. Res. Commun. 136, 80-85. 17. Estrada, G., et al. (1996). Mol. Cell Probe 10, 179-185. 52. Schuberth, H.J., et al. (1996). J. Immunol. Methods 18. Fouassier, L., et al. (2000). J. Biol. Chem. 275, 25039- 189, 89-98. 25045. 53. Schumacher, T.N., et al. (1996). Science 271, 1854- 19. Gao, C., et al. (1997). Proc. Natl. Acad. Sci. U.S.A. 94, 1857. 11777-11782. 54. Schwarzman, A.L., et al. (1999). Proc. Natl. Acad. Sci. 20. Geselowitz, D.A. and Neumann, R.D., (1995). U.S.A. 96, 7932-7937. Bioconjugate Chem. 6, 502-506. 55. Scott, M.G., et al. (2000). J. Immunol. 164, 549-553. 21. Glover, B.P. and McHenry, C.S., (2001). Cell 105, 925- 934. 56. Sharma, K.K., et al. (2000). J. Biol. Chem. 275, 3767- 3771. 22. Guo, Y., et al. (2001). J. Biol. Chem. 276, 45791-45799. 57. Singh, Y., et al. (1999). Infect. Immun. 67, 1853-1859. 23 Hiller, Y., et al. (1987). Biochem. J. 248, 67-171. 58. Slatin, S.L., et al. (2002). Proc. Natl. Acad. Sci. U.S.A. 24. Hofman, K., et al. (1980). Proc. Natl. Acad. Sci. U.S.A. 99, 1286-1291. 77, 4666-4668. 59. Smith, J.S., et al. (1999). Proc. Natl. Acad. Sci. U.S.A. 25. Horney, Mark J., et al. (2001). J. Biol. Chem. 276, 2880- 96, 8855-8860. 2889. 60. Sutoh, K., et al. (1984). J. Mol. Biol. 178, 323-339. 26 Huh, K-H. and Wenthold, R.J. (1999). J. Biol. Chem. 274, 151-157. 61. Tang, A., et al. (1993). Nature 361, 82-85. 27. Ilver, D., et al. (1998). Science 279, 373-377. 62. Trotti, D., et al. (2001). J. Biol. Chem. 276, 576-582. 28. Ishmael, F.T., et al. (2002). J. Biol. Chem. 277, 20555- 63. Trotman, L.C., et al. (2001). Nat. Cell Biol. 3, 1092-1100. 20562. 64. Wassarman, D.A. (1993). Mol. Biol. Rep. 17, 143-151. 29. Ishmael, F.T., et al. (2001). J. Biol. Chem. 276, 25236- 65. Wilchek, M. and Bayer, E.A. (1989). Protein Recognition 25242. of Immobilized Ligands. Hutchins, T.W., ed. Alan R. 30. Jacobson, et al. (1995). Life Sci. 56, 823-830. Liss, Inc., pp. 83-90. 31. Ju, T., et al. (2002). J. Biol. Chem. 277, 169-177. 66. Yamamoto, K., et al. (1984). FEBS Lett. 176, 75-78. 32. Kahne, T. and Ansorge, S. (1994). J. Immunol. Methods 67. Yu, Q. and Toole, B.P. (1995). Biotechniques 19, 122- 168, 209-218. 129. 33. Kilic, F., et al. (2000). Proc. Natl. Acad. Sci. U.S.A. 97, 68. Zeheb, R., et al. (1983). Anal. Biochem. 129, 156-161. 3106-3111. 69. Zhang, L., et al. (1999). J. Biol. Chem. 274, 8966-8972. 34. Kim, K., et al. (2001). J. Biol. Chem. 276, 40591-40598. 70. Zuk, P.A. and Elferink, L.A. (2000). J. Biol. Chem. 275, 35. Kleene, R., et al. (2000). Biochemistry 39, 9893-9900. 26754-26764. 36. Lacey, E. and Grant, W.N., (1987). Anal. Biochem. 163, 151-158.

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© Pierce Biotechnology, Inc., 2005. Printed in the U.S.A. Pierce products are supplied for laboratory or manufacturing applications only. Prices listed herein are accurate at time of printing. For up-to-date pricing, visit our web site. # 1601227 06/05 *Slide-A-Lyzer® Dialysis Cassette Technology is protected by U.S. Patent # 5,503,741.