title: Activation, Isolation, and Analysis of the Death-Inducing Signaling Complex.
abstract: This protocol describes activation, isolation, and analysis of the CD95 (APO-1/Fas) death-inducing signaling complex (DISC) using affinity purification. Activation is achieved using a biotin-labeled anti-CD95 antibody and the native DISC complex is captured using streptavidin beads. This approach minimizes both the number of steps involved and any potential nonspecific interactions or cross-reactivity of antibodies commonly seen in immunoprecipitations using unlabeled antibodies and protein A/G beads. Composition of the isolated complex is analyzed via western blot to identify known DISC components, and dimerization-induced autocatalytic processing of procaspase-8 at the DISC can be confirmed by detection of caspase-8 cleavage products. The potential for DISC-associated caspase-8 to activate the caspase cascade can be determined by measuring caspase-8-dependent cleavage of the fluorigenic substrate Ac-IETD.AFC, or by performing a bioassay using exogenous protein substrates.
authors.affiliation: MRC Toxicology Unit, Hodgkin Building, Leicester, LE1 9HN, United Kingdom.
authors.forename: Michelle A
authors.initials: MA
authors.lastname: Hughes
authors.affiliation: MRC Toxicology Unit, Hodgkin Building, Leicester, LE1 9HN, United Kingdom.
authors.forename: Claudia
authors.initials: C
authors.lastname: Langlais
authors.affiliation: MRC Toxicology Unit, Hodgkin Building, Leicester, LE1 9HN, United Kingdom.
authors.forename: Kelvin
authors.initials: K
authors.lastname: Cain
authors.affiliation: MRC Toxicology Unit, Hodgkin Building, Leicester, LE1 9HN, United Kingdom.
authors.forename: Marion
authors.initials: M
authors.lastname: MacFarlane
mesh_terms: D017209:Apoptosis; D001323:Autoantibodies / Q000378:metabolism; D001710:Biotin / Q000378:metabolism; D015153:Blotting, Western; D002846:Chromatography, Affinity; D053418:Death Domain Receptor Signaling Adaptor Proteins / Q000032:analysis*; D006801:Humans; D019169:Jurkat Cells; D008863:Microspheres; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D015398:Signal Transduction; D013194:Staining and Labeling; D019809:Streptavidin / Q000378:metabolism; D019014:fas Receptor / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D001323:Autoantibodies; D053418:Death Domain Receptor Signaling Adaptor Proteins; C491901:FAS protein, human; D009097:Multienzyme Complexes; D019014:fas Receptor; D001710:Biotin; D019809:Streptavidin