Immunoprecipitation of Death Inducing Signaling Complex by Caspase-8

Andrei Alexandru Constantinescu (1, 2) , Aymeric Morlé (1, 2) , Olivier Micheau (1, 2)

1 LabEx LipSTIC - Laboratoire d'Excellence : Lipoprotéines et Santé : prévention et Traitement des maladies Inflammatoires et du Cancer
2 LNC - Lipides - Nutrition - Cancer [Dijon - U1231]

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.

Mots clés: Immunoprecipitation; Caspase-8; DISC; Electrophoresis; Immunoblots; Protein extracts; Caspase-8; DISC; Electrophoresis; Immunoblots; Protein extracts

Domaines: Immunologie

Andrei Alexandru Constantinescu, Aymeric Morlé, Olivier Micheau. Immunoprecipitation of Death Inducing Signaling Complex by Caspase-8. Springer Protocols. CD 95 : Methods and Protocols, 1557, Humana Press, pp.19-31, 2017, 978-1493967780. ⟨10.1007/978-1-4939-6780-3_22⟩. ⟨hal-01548294⟩