Glossary
Biotin Label
Biotinylation equips a peptide with a femtomolar-affinity handle for streptavidin capture. Spacer length, endogenous biotin and elution limits in practice.
A biotin tag is biotin (vitamin B7) chemically coupled to a peptide, usually at the N-terminus or on the side chain of a lysine. What makes it useful is its binding to streptavidin: the dissociation constant lies in the femtomolar range, making it one of the tightest non-covalent interactions known in biology, and it holds up against detergents, high salt and moderate denaturants.
Applications and practical limits
Biotin-tagged peptides are the go-to reagent for pull-down experiments and target identification using streptavidin magnetic beads, for anchoring on streptavidin-coated ELISA plates and SPR chips, and for signal-amplified detection in which a streptavidin-HRP conjugate takes the place of a labelled secondary antibody.
Three limitations come up repeatedly. The first is spacer length: biotin’s valeric acid chain is short while the binding pocket is deep, so an aminohexanoic acid or PEG spacer is usually added to keep the peptide within reach. The second is endogenous biotin: liver, kidney, brain and similar tissues hold enough free biotin to compete, so an avidin/biotin blocking step becomes necessary. The third is elution: the bond is so tight that intact complexes can generally only be recovered by boiling in SDS, by long incubation with excess free biotin, or through a cleavable linker planned into the design from the outset.
Related terms and material
FAM/FITC label · I-125 radiolabel · purity. Reference material: labeled peptides, labeled antibodies. Further reading: labeled peptides guide.