Exploration of Fas S-Nitrosylation by the Biotin Switch Assay

: S-nitrosylation is the covalent attachment of nitric oxide radical to the thiol side chain of cysteine. The death receptor Fas/CD95 can be S-nitrosylated in cancer cell lines by NO donors or iNOS activation. This posttranslational modification (PTM) induces Fas aggregation into lipid rafts and enhances FasL-mediated signaling and apoptosis. In this report, we describe the detection of Fas S-nitrosylation by the most commonly used method, the biotin switch assay (BSA) technique, that allows the detection of this very labile covalent modification in cells or tissues. Briefly, this technique relies on the ability of ascorbate to reduce the covalent bond between the NO radical and the protein, allowing the exchange of the NO radical with a thiol reactive biotin-HPDP. The biotinylated proteins are then easily purified by using NeutrAvidin resin, separated by SDS-PAGE resolution and analyzed by Western blotting.