Biotin peptide is the common name for a small molecule that can be used to label proteins in a similar manner as DIG (dithiothreitol) or FITC (fluorescein isothiocyanate). In contrast to enzymatic methods, chemical biotinylation can be performed both in vitro and in vivo. It is also more versatile in the type of sulfhydryl-reactive groups it can introduce and provides greater flexibility in protein modification than enzymatic methods.
The high affinity interaction between biotin and streptavidin or avidin (KD
A number of natural and synthetic peptide sequences have been enzymatically biotinylated in vivo to serve as a protein-specific affinity tag. These peptides are usually conjugated to a larger carrier protein such as bovine serum albumin (BSA), ovalbumin or keyhole limpet hemocyanin (KLH). They can then be captured by immobilization on streptavidin-coated beads, membranes or microtiter plates using competitive displacement with free biotin or monomeric avidin/streptavidin under mild conditions.
Most commonly, biotinylation is carried out in the peptide by inserting one cysteine residue into the peptide acceptor domain that can be coupled to a carrier protein through maleimide or pyridyl disulfide. Maleimide-type reagents like BMCC-Biotin and iodoacetyls are highly reactive towards the sulfhydryl group of the target peptide, but require acidic to neutral pH. Pyridyl disulfide-based biotinylation reagents such as Thermo Scientific EZ-Link HPDP-Biotin are highly reactive towards the sulfhydryl groups of a peptide, but require a higher pH (pH 7.5-8.5). In order to avoid sterical hindrance between the target peptide and the biotin, a hydrophobic straight chain spacer such as GG or an aminohexanoic acid tetrapeptide can be inserted into the peptide acceptor.