Simplified overview of the biotin switch assay for detecting S-acylated proteins. For simplicity only four proteins are shown in this scheme, two S-acylated, and two not. In reality all of the proteins from a lysate are subjected to this treatment resulting in the purification of all S-acylated proteins in a sample. (1) Cell lysates containing proteins (black lines) with cysteine residues (open circles) may have acyl groups (red lines) attached through a thioester bond (S-acylation). (2) Lysates are treated with the sulfhydryl reactive reagent N-ethylmaleimide (NEM, blue line) to block free cysteines. (3) The sample is then divided into 2 equal portions and one treated with the S-acyl group cleavage reagent hydroxylamine (Hyd+) and one without (Hyd-). Both samples are then treated with sulfhydryl reactive biotin (green line). Free sulfhydryls liberated by hydroxylamine treatment (Hyd+) are labelled with sulfhydryl reactive biotin to form a biotinylated cysteine residue. Samples incubated in the absence of hydroxylamine (Hyd-) do not undergo biotinylation as free sulfhydryls are not generated. (4) Following biotinylation a sample of each reaction is removed to act as a column loading control (C Hyd+ and C Hyd-). The remaining sample is loaded onto neutravidin beads and biotinylated proteins, representing S-acylated proteins in the lysate from step 1, are purified (E Hyd+ and E Hyd-). (5) After elution from the neutravidin beads protein samples are separated by SDS-PAGE and the protein of interest (POI) can be assayed for S-acylation by western blot using an anti-POI antibody. The pattern of signals expected of an S-acylated protein assayed by this method is shown. Proteins that were not S-acylated would produce a signal from C Hyd+ and C Hyd- samples but not from E Hyd+ and E Hyd- samples. Proteins that were biotinylated in planta, rather than as a result of the biotin switch protocol, would produce a signal in both the E Hyd+ and E Hyd- samples. In these cases the level of in planta biotinylation will be evident from the E Hyd- lane.