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CD95 pp 189-198 | Cite as

Detection of S-Acylated CD95 by Acyl-Biotin Exchange

  • Aurelie Rossin
  • Anne-Odile HueberEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1557)

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.

Key words

S-acylation S-palmitoylation Hydroxylamine Acyl-biotin exchange 

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Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  1. 1.Institut de Biologie Valrose, CNRS UMR 7277, INSERM UMR 1091Université Côte d’AzurNiceFrance
  2. 2.Institut de Biologie Valrose, CNRS UMR 7277, INSERM UMR 1091,Université de NiceNiceFrance

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