Sulfhydryl groups are the second most common target for modification in biological studies. They are found as exposed cysteines in protein and peptides. Sulfhydryl group biotinylation provides an advantage in some applications; for example, targeting a sulfhydryl group can help preserve the biological activity of an enzyme when amines are found at the active site.

Maleimide-PEG₂ and PEG₁₁-Biotin: Water-soluble. Reacts with sulfhydryl (-SH) groups at acidic to neutral pH. PEG₁₁-Biotin has an extra long spacer arm that reduces steric hindrance.

Biotin-BMCC: Reacts with sulfhydryl (-SH) groups at 6,5 to 7,5 pH. Avoids potential modification of tyrosine residues that can be associated with Iodoacetyl-Biotin. Must be dissolved in DMSO or DMF before use.

Iodoacetyl-PEG₂-Biotin: Water soluble. Reacts with sulfhydryl (-SH) groups at basic to neutral pH. Coupling reactions occur in the dark.

Iodoacetyl-LC-Biotin: Reacts with sulfhydryl (-SH) groups at basic to neutral pH. Coupling reactions occur in the dark. Must be dissolved in DMSO or DMF before use.

Biotin-HPDP: Pyridyldithiol reacts with sulfhydryl (-SH) groups to form a stable disulfide bond between pH 6 to 9. Pyridine-2-thione leaving group can be used to measure degree of biotinylation at 343 nm. Disulfide bond is cleavable using 50 mM DTT or 100 mM 2-mercaptoethanol. Must be dissolved in DMSO or DMF before use.