The affinity between avidin and biotin is one of the strongest known non-covalent interactions of a protein and ligand (Ka=1015M-1). This allows molecules with biotin attachment to be bound with avidin conjugates. The bond formation is rapid. It is stable even with extreme temperature, pH , denaturing agents and organic solvents. 


Biotinylated proteins can be used for detection in enzyme-linked immunosorbant assay (ELISA), Western blot analysis, immunohistochemistry (IHC), immunoprecipitation (IP), cell surface labeling and flow cytometry/fluorescence-activated cell sorting (FACS). They also can be used to facilitate protein purification with avidin conjugates.


Comparing with the size of protein, biotin is much smaller, which makes the interference of labeling minimal. The valeric acid side chain is easily derivatized and conjugate to reactive moieties on protein.

 Labeling methods

Biotinylation can be performed both by enzymatic, via co-expression of bacterial biotin ligase and modified protein with a biotin acceptor peptide, or chemical means (in vitro and in vivo). Although enzymatic methods offer better uniform labeling, chemical labeling is still the most widely used methods because of the great flexibility.

 Key features of biotinylation reagents

Reactive Moiety—Spacer Arm—Biotin. The variation between these features determines the characteristic of the reagents. For example, longer Spacer Arm can increase the availability of biotin for avidin binding. When choosing biotin labeling reagents, the following characteristic should be considered.

  • Solubility - hydrophobic or hydrophilic protein
  • Spacer arm - availability of biotin for avidin binding
  • Cleavability/reversibility - cleavability of biotin to facilitate purification
  • Functional group - reactive moieties for nonselective or targeted biotinylation.