Biotinylation of Proteins

biotin peptide

The high binding affinity of streptavidin for biotin and its ability to detect several ligands simultaneously makes it an ideal tagger for identifying protein function. However, the biotin-(streptavidin)avidin interaction is essentially irreversible under physiological conditions and susceptible to acid and/or temperature changes as well as organic solutions and denaturing agents.

In order to overcome this limitation, a number of different chemical biotinylation methods have been developed that utilize reversible interactions. The most commonly used reagents use the amino group of an exogenous protein (such as a cell-free expression system, peptide, or antibody) to react with the carboxylate group on biotin. This enables the proteins to be eluted from the streptavidin-biotin affinity resin under mild conditions based on competitive displacement with free biotin rather than harsh elution conditions that could disassociate or damage the protein.

Biotin-biotinylation reactions are most commonly performed through chemical means, although enzymatic approaches can also be employed. Both chemical and enzymatic methods can be performed at room temperature with minimal instrumentation, but the use of chemical reagents generally provides more flexibility in the type of biotinylation needed for a given application.

The chemical reagents available for biotinylation of proteins include maleimide, BMCC-biotin, and haloacetyl groups. These are reactive towards sulfhydryl groups at acidic to neutral pH, which is optimal for protein-protein crosslinking reactions with streptavidin. Each of these reagents is available with a variety of spacer arms, from 2 to 14 adeno groups, that can be varied to accommodate varying distances between the biotin and the protein-binding site.


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