title: Site-Specific Detection of Tyrosine Phosphorylated CD95 Following Protein Separation by Conventional and Phospho-Protein Affinity SDS-PAGE.
abstract: Phosphorylation of two tyrosines in the death domain of CD95 is a critical mechanism in determining the receptor's choices between cell death and survival signals. Recently, site-specific monoclonal antibodies against phosphorylated tyrosines of CD95 have been generated and used to successfully detect each phosphorylated death domain tyrosine of CD95 directly and separately by immunoblotting. Here we provide detailed protocols and useful tips for a successful site-specific detection of phosphorylated death domain tyrosine of CD95 following a protein separation by sizes (conventional SDS-PAGE) and by degrees of phosphorylation (phospho-protein affinity, mobility shift SDS-PAGE).
authors.affiliation: Université Côte d'Azur, Institut de Biologie Valrose, CNRS UMR 7277, Inserm UMR 1091, Parc Valrose, Bâtiment des Sciences Naturelles, 06108, Nice, France.
authors.forename: Krittalak
authors.initials: K
authors.lastname: Chakrabandhu
authors.affiliation: Université Côte d'Azur, Institut de Biologie Valrose, CNRS UMR 7277, Inserm UMR 1091, Parc Valrose, Bâtiment des Sciences Naturelles, 06108, Nice, France.
authors.forename: Sébastien
authors.initials: S
authors.lastname: Huault
authors.affiliation: Institut de Biologie Valrose, CNRS UMR 7277, INSERM UMR,Université de Nice, Nice, France. hueber@unice.fr.
authors.forename: Anne-Odile
authors.initials: AO
authors.lastname: Hueber
mesh_terms: D002460:Cell Line; D002846:Chromatography, Affinity / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D024202:Electrophoretic Mobility Shift Assay; D006801:Humans; D015151:Immunoblotting; D010750:Phosphoproteins / Q000737:chemistry / Q000302:isolation & purification*; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000737:chemistry / Q000378:metabolism*; D019014:fas Receptor / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*
chemical_list: D010750:Phosphoproteins; D019014:fas Receptor; D019000:Phosphotyrosine
keywords: CD95; Immunoblot; Mobility shift; Phos-tag™; Phosphorylation; SDS-PAGE