The highly specific interaction of avidin with biotin (vitamin H) can be a useful tool in designing nonradioactive purification and detection systems. The extraordinary affinity of avidin for biotin (Ka = 10)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-hydroxysulfosuccinimide (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.
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