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The Biotin HPDP is a membrane permeable biotin labeling reagent that reacts with sulfhydryl (-SH) groups. The resulting disulfide bond between the target sulfhydryl molecule and the biotin group can be cleaved by reducing agents to release the biotin group and regenerate the protein (or peptide) in its original, unmodified form (Figure 1). Labeling with Biotin HPDP is convenient when using immobilized avidin or streptavidin to purify the target molecules for reducing SDS-PAGE or mass analysis; the captured biotinylated molecules can be efficiently eluted from the support by cleaving the disulfide bond with dithiothreitol (DTT) or other reducing agent rather than by attempting to dissociate the high affinity interaction between avidin and biotin with strong acid or denaturant.
HPDP-Biotin is not soluble in aqueous buffer; it must be dissolved in organic solvent before addition to an aqueous reaction. Use dimethylsulfoxide (DMSO) or dimethylformamide (DMF).
The 2-pyridyldithio group of HPDP-Biotin reacts optimally with free (reduced) sulfhydryls at pH 7-8. Reaction buffers must be free of thiols and disulfide reducing agents until quenching or reduction of the 2-pyridyldithiol is desired.