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Methods Mol Biol. 2017;1557:189-198. doi: 10.1007/978-1-4939-6780-3_17.

Detection of S-Acylated CD95 by Acyl-Biotin Exchange.

Author information

1
Institut de Biologie Valrose, CNRS UMR 7277, INSERM UMR 1091, Université Côte d'Azur, Parc Valrose, Bâtiment des Sciences Naturelles, 06108, Nice, France.
2
Institut de Biologie Valrose, CNRS UMR 7277, INSERM UMR 1091,Université de Nice, Nice, France. hueber@unice.fr.

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.

KEYWORDS:

Acyl-biotin exchange; Hydroxylamine; S-acylation; S-palmitoylation

PMID:
28078593
DOI:
10.1007/978-1-4939-6780-3_17
[Indexed for MEDLINE]

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