Biotinylating

Biotinylating is the process of covalently attaching biotin to a molecule, most commonly proteins, peptides, nucleic acids, or small molecules, so that the biotinylated product can be captured or detected through the strong interaction with streptavidin or avidin.

Chemical biotinylation typically uses reactive biotin derivatives such as N-hydroxysuccinimide (NHS) esters (for example NHS-biotin, sulfo-NHS-biotin). These reagents react with primary amines, primarily the lysine side chains and N-termini of proteins, to form stable amide bonds. Reactions are conducted under mildly basic conditions and parameters such as reagent excess, pH, and time control the labeling density. After labeling, unreacted biotin is removed by dialysis or chromatography. The degree of labeling is assessed by spectroscopic methods or colorimetric assays and reported as biotin groups per molecule or per reaction.

Enzymatic biotinylation offers a more uniform alternative. E. coli biotin ligase (BirA) catalyzes the transfer of biotin to a specific lysine within a short recognition sequence, commonly the AviTag. This site-specific approach yields homogeneous biotinylation with less risk of compromising protein function. It often requires biotin, ATP, and the BirA enzyme in defined conditions.

Applications of biotinylated molecules include purification and detection on streptavidin- or avidin-coated surfaces, pull-down assays, immunoassays, Western blotting, ELISA, and imaging. Biotinylation is widely used in affinity capture, labeling of biomolecules for sensors, and in methods requiring strong, specific coupling. Considerations include the potential impact on activity or binding, labeling density, and ensuring removal of excess biotin or reagents.