In Vitro Assembly and Analysis of the Apoptosome Complex
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Updated: Feb 14, 2026

Analysis of the Expression and Complexes Assembly of the Mitochondrial Respiratory Chain Proteins in the Fission Yeast Schizosaccharomyces pombe
Published on: May 2, 2025
Claudia Langlais1, Michelle A Hughes1, Kelvin Cain1
1MRC Toxicology Unit, Hodgkin Building, Leicester LE1 9HN, United Kingdom.
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.
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