Fig. 4.

Schematic representation of the acyl biotin exchange reaction. Acyl biotin exchange relies on the sensitivity to hydroxylamine (NH₂OH) treatment of the covalent thioester linkage between palmitic acid moieties and cysteine residues of protein substrates. In the first step, parasite lysates are treated with N-ethylmaleimide to block reactive free thiols. The sample is then split in half and incubated with the thiol-labeling reagent biotin-HPDP in the presence or absence of NH₂OH. Liberation of thiols by NH₂OH cleavage enables specific labeling, while the other half of the sample serves as a control for nonspecific labeling due to incomplete preblock, or later, nonspecific purification during streptavidin chromatography.