Detection of S-Acylated CD95 by Acyl-Biotin Exchange

摘要: S-acylation is the covalent addition of a fatty acid, most generally palmitate onto cysteine residues proteins through labile thioester linkage. The death receptor CD95 S-palmitoylated and this post-translational modification plays crucial role on organization in cellular membranes thus CD95-mediated signaling. Here, we describe nonradioactive detection by acyl-biotin exchange chemistry which biotin substituted for CD95-linked acid. This sensitive technique, depends ability hydroxylamine to specifically cleave linkage between acids proteins, relies three chemical steps: (1) blockage free thiols non-modified residues, (2) hydroxylamine-mediated cleavage thioester-linked restore (3) biotinylation with thiol reactive agent. Resulting biotinylated can be easily purified an avidin capture analyzed SDS-PAGE immunoblotting.