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

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

  • STAR Protoc. 2024 May 4;5(2):103054. doi: 10.1016/j.xpro.2024.103054.
Stuart Leishman 1,  Najd M Aljadeed 2,  Paras K Anand 3
Affiliations

Affiliations

  • 1 Department of Infectious Disease, Imperial College London, London W12 0NN, UK. Electronic address: s.leishman21@imperial.ac.uk.
  • 2 Department of Infectious Disease, Imperial College London, London W12 0NN, UK.
  • 3 Department of Infectious Disease, Imperial College London, London W12 0NN, UK. Electronic address: paras.anand@imperial.ac.uk.
PMID: 38704832 DOI: 10.1016/j.xpro.2024.103054
Abstract

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.

Keywords

cell biology; immunology; protein biochemistry.

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