Biotinylating Antibodies Using Biotin Polyethylene Oxide (PEO) Iodoacetamide

There are several techniques for biotinylating antibodies, from the most basic (using NHS-ester biotin to label primary amines) to more complex experiments (modifying sulfhydryls and carbohydrates). Biotinylation of free sulfhydryls, described here, can be effectively mediated using haloacetyl biotin derivatives. To modify an antibody using this reagent, sulfhydryls must be available. Digestion of antibodies by the enzyme pepsin produces F(ab')₂ fragments, which can be separated by mild reduction into two sulfhydryl-containing, univalent Fab' fragments. Alternatively, thiol groups can be added by modifying amines with an appropriate cross-linker.