Exploration of Fas S-Nitrosylation by the Biotin Switch Assay

Abstract : 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.
Type de document :
Chapitre d'ouvrage
Liste complète des métadonnées

https://hal-univ-bourgogne.archives-ouvertes.fr/hal-01490243
Contributeur : Liic - Université de Bourgogne <>
Soumis le : mercredi 15 mars 2017 - 10:06:00
Dernière modification le : mercredi 5 septembre 2018 - 17:04:03

Identifiants

Citation

Ali Bettaieb, Catherine Paul, Stéphanie Plenchette. Exploration of Fas S-Nitrosylation by the Biotin Switch Assay. CD95 : Methods and Protocols , 1557, Springer New York, pp.199-206, 2017, Methods in Molecular Biology, 978-1-4939-6778-0 (Print) 978-1-4939-6780-3 (Online). ⟨10.1007/978-1-4939-6780-3_18⟩. ⟨http://link.springer.com/protocol/10.1007/978-1-4939-6780-3_18⟩. ⟨hal-01490243⟩

Partager

Métriques

Consultations de la notice

75