Verification of a Putative Caspase Substrate

Abstract

Proteomic approaches have been adopted to survey the degradome of caspases during apoptosis. These approaches provide a comprehensive list of substrates and give clues to which pathways are altered during apoptosis by activated caspases. However, substrates identified by large-scale proteomic screening need to be validated as bona fide caspase targets. This ensures that conclusions derived from the screen are based on real substrates and not on artifacts of the proteomic screen. The validation method described in this protocol uses radiolabeled versions of the putative substrates synthesized using in vitro transcription/translation methods. These are incubated with purified caspases to determine whether they are genuine caspase substrates.