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RESEARCH PRODUCT
Exploration of Fas S-Nitrosylation by the Biotin Switch Assay
Catherine PaulCatherine PaulAli BettaiebAli BettaiebStéphanie PlenchetteStéphanie Plenchettesubject
0301 basic medicineBiotin switch assaybiologyChemistryNitrosylationNeutrAvidinNitric oxideS-NitrosylationFas receptorGlyceryl trinitrate3. Good health03 medical and health sciences030104 developmental biology0302 clinical medicineBiochemistryApoptosisCovalent bondFas S030220 oncology & carcinogenesisBiotinylationbiology.protein[SDV.BBM]Life Sciences [q-bio]/Biochemistry Molecular BiologyLipid raft[ SDV.BBM ] Life Sciences [q-bio]/Biochemistry Molecular BiologyCysteinedescription
International audience; 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.
| year | journal | country | edition | language |
|---|---|---|---|---|
| 2017-01-01 |