Protocol for a semi-quantitative approach to identify protein S-palmitoylation in cultured cells by acyl biotin exchange assay

Palmitoylation is a post-translational lipid modification in which palmitic acid is conjugated predominantly to cysteine residues of target proteins, allowing them to tether to cell membranes. Here, we describe a protocol to perform a stepwise acyl biotin exchange assay to identify protein S-palmitoylation. We describe steps for initial blocking of free thiols in protein lysates, subsequent replacement of thioester-linked palmitate groups with a biotin tag for affinity enrichment, and identification of palmitoylated proteins by SDS-PAGE. For complete details on the use and execution of this protocol, please refer to Leishman et al.¹.