acylRAC

Acyl-RAC, short for acyl-resin-assisted capture, is a biochemical method used to profile protein S-acylation, a reversible post-translational modification in which fatty acyl groups, commonly palmitate, are covalently attached to cysteine residues via thioester bonds. The technique provides enrichment and identification of acylated proteins, facilitating downstream analysis by Western blotting or mass spectrometry.

The general workflow begins with sample preparation and blocking of free thiols to prevent non-specific labeling, typically using reagents such as N-ethylmaleimide or iodoacetamide. After blocking, thioester bonds bearing acyl groups are selectively cleaved by hydroxylamine, exposing new free cysteine thiols that were previously acylated. These newly generated thiols readily react with a thiol-reactive resin (for example, disulfide- or sulfhydryl-reactive media) to capture the formerly acylated proteins. Following stringent washing, bound proteins are eluted by reducing conditions and analyzed by downstream methods such as SDS-PAGE and immunoblotting or identified by mass spectrometry. The approach commonly uses a resin that forms a reversible disulfide linkage to enable gentle elution.

Applications of acyl-RAC include mapping S-palmitoylation across proteomes, studying dynamics of palmitoylation in signaling and trafficking pathways, and comparing acylation patterns in different cell types or disease states. It is often used alongside complementary methods, such as acyl-PEG exchange or metabolic labeling, to corroborate palmitoylation data.

Limitations include potential incomplete thiol blocking, non-specific hydrolysis of other thioester bonds, and the need for appropriate controls to distinguish true acylation from artifacts. Negative controls without hydroxylamine and validation by independent methods are recommended.