Chapter title |
Detection of S-Acylated CD95 by Acyl-Biotin Exchange
|
---|---|
Chapter number | 17 |
Book title |
CD95
|
Published in |
Methods in molecular biology, January 2017
|
DOI | 10.1007/978-1-4939-6780-3_17 |
Pubmed ID | |
Book ISBNs |
978-1-4939-6778-0, 978-1-4939-6780-3
|
Authors |
Aurelie Rossin, Anne-Odile Hueber |
Editors |
Patrick Legembre |
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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