Abstract
S-acylation is the covalent addition of a fatty acid, most generally palmitate onto cysteine residues of proteins through a labile thioester linkage. The death receptor CD95 is S-palmitoylated and this post-translational modification plays a crucial role on CD95 organization in cellular membranes and thus on CD95-mediated signaling. Here, we describe the nonradioactive detection of CD95 S-acylation by acyl-biotin exchange chemistry in which a biotin is substituted for the CD95-linked fatty acid. This sensitive technique, which depends on the ability of hydroxylamine to specifically cleave the thioester linkage between fatty acids and proteins, relies on three chemical steps: (1) blockage of free thiols of non-modified cysteine residues, (2) hydroxylamine-mediated cleavage of thioester-linked fatty acids to restore free thiols and (3) biotinylation of free thiols with a thiol reactive biotinylation agent. Resulting biotinylated proteins can be easily purified by an avidin capture and analyzed by SDS-PAGE and immunoblotting.
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Rossin, A., Hueber, AO. (2017). Detection of S-Acylated CD95 by Acyl-Biotin Exchange. In: Legembre, P. (eds) CD95. Methods in Molecular Biology, vol 1557. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6780-3_17
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DOI: https://doi.org/10.1007/978-1-4939-6780-3_17
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