Biotinylation of proteins is a powerful tool for investigating biological phenomenon, both in vitro and in vivo. Biotinylating reagents that form covalent bonds with several types of amino acid residues are commercially available. However, most, if not all, of these commercially available biotinylating agents produce biotin–protein bonds that are susceptible to cleavage in human plasma. Here, we describe the use of immunoglobulin G as a model protein for evaluation of biotin–protein bond stability and for the investigation of the mechanism of biotin release. We also describe the synthesis of a biotin–protein bond that is stable in human plasma and a method for evaluation of that stability.
