title: In Vitro Assembly and Analysis of the Apoptosome Complex.
abstract: This protocol describes an in vitro model for studying the mechanisms of caspase activation and native apoptosome complex assembly in cell-free extracts. Active caspases in dATP-activated lysates are detected by fluorimetry using a tetrapeptide substrate (DEVD) tagged with a fluorophore (AFC), which, when released, produces a real-time readout for caspase-3 and -7 (DEVDase) activity. Gel filtration is used to isolate the apoptosome complex from the activated lysates, and assembly of Apaf-1 and caspase-9 from their monomeric forms into the multiprotein apoptosome can be confirmed via western blot. Apoptosome complex activity can be shown by incubation with exogenous procaspase-3 and -7 followed by fluorimetric bioassay (to confirm functionality of the processed effector caspases) and/or western blotting (for detection of cleaved caspase-3 and -7). A method for preparation of free procaspases for the bioassay is also described.
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: Michelle A
authors.initials: MA
authors.lastname: Hughes
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: D000818:Animals; D053478:Apoptosomes / Q000737:chemistry* / Q000302:isolation & purification*; D015153:Blotting, Western; D020169:Caspases / Q000032:analysis; D002460:Cell Line; D002474:Cell-Free System; D002850:Chromatography, Gel; D005470:Fluorometry; D006801:Humans; D051381:Rats
chemical_list: D053478:Apoptosomes; D020169:Caspases