title: Immunoprecipitation of Death Inducing Signaling Complex by Caspase-8.
abstract: Analysis of CD95/Fas complexes by immunoprecipitation has long relied on the monoclonal antibody APO1 or tagged recombinant Fas ligand. Immunoprecipitation is an elegant and efficient procedure to investigate endogenous protein interactions or complexes. Provided that the targeted complex is soluble in mild detergent these complexes can be recovered using protein A/G-coupled Sepharose beads and further analyzed after denaturation and electrophoretic separation by western blotting or mass spectrometry. Herein, we describe in detail the method used in our laboratory to immunoprecipitate and analyze by immunoblot complexes containing caspase-8, using a commercial antibody directed against caspase-8.
authors.affiliation: INSERM, UMR866, "Equipe labellisée Ligue contre le Cancer" and Laboratoire d'Excellence LipSTIC, Dijon, 21079, France.|UFR Sciences de Santé, Univ. Bourgogne, Dijon, 21079, France.
authors.forename: Andrei Alexandru
authors.initials: AA
authors.lastname: Constantinescu
authors.affiliation: INSERM, UMR866, "Equipe labellisée Ligue contre le Cancer" and Laboratoire d'Excellence LipSTIC, Dijon, 21079, France.|UFR Sciences de Santé, Univ. Bourgogne, Dijon, 21079, France.
authors.forename: Aymeric
authors.initials: A
authors.lastname: Morlé
authors.affiliation: INSERM, UMR866, "Equipe labellisée Ligue contre le Cancer" and Laboratoire d'Excellence LipSTIC, Dijon, 21079, France. omicheau@u-bourgogne.fr.|UFR Sciences de Santé, Univ. Bourgogne, Dijon, 21079, France. omicheau@u-bourgogne.fr.
authors.forename: Olivier
authors.initials: O
authors.lastname: Micheau
mesh_terms: D015153:Blotting, Western; D053181:Caspase 8 / Q000378:metabolism*; D053418:Death Domain Receptor Signaling Adaptor Proteins / Q000378:metabolism*; D053222:Fas Ligand Protein / Q000378:metabolism; D047468:Immunoprecipitation / Q000379:methods; D046912:Multiprotein Complexes / Q000378:metabolism; D011485:Protein Binding; D019014:fas Receptor / Q000378:metabolism
chemical_list: D053418:Death Domain Receptor Signaling Adaptor Proteins; D053222:Fas Ligand Protein; D046912:Multiprotein Complexes; D019014:fas Receptor; D053181:Caspase 8
keywords: Caspase-8; DISC; Electrophoresis; Immunoblots; Immunoprecipitation; Protein extracts