title: Assaying caspase activity in vitro.
abstract: Monitoring the activity of a caspase, either as an isolated protein or in a complex mixture (e.g., a cytosolic extract), can be achieved by measuring substrate cleavage. Chromogenic or fluorogenic substrates are available for many caspases. These substrates usually consist of the four-amino-acid motif that is optimal for each caspase and a moiety that, when cleaved, generates either a chromophore or a fluorophore that can be detected using spectrophotometric or fluorimetric means. In this protocol, we describe how to use these substrates to monitor caspase activity in samples containing active caspases (e.g., apoptotic cells). Caspase inhibitors, which contain a moiety that covalently attaches to the active site of the caspase, can be used in these assays. These assays will ascertain whether caspases are involved in a specific process (e.g., whether caspases are activated after an apoptotic stimulus) and are particularly informative if a purified caspase is used. However, the substrates and inhibitors are not specific for a particular caspase in an environment containing multiple caspases. So, if cytosolic or apoptotic cell extracts are used in these assays, additional experiments must be performed to identify exactly which caspases are involved.
authors.affiliation: Department of Life Sciences, New York Institute of Technology, Old Westbury, New York 11568;
authors.forename: Gavin P
authors.initials: GP
authors.lastname: McStay
authors.affiliation: Department of Immunology, St. Jude Children's Research Hospital, Memphis, Tennessee 38105.
authors.forename: Douglas R
authors.initials: DR
authors.lastname: Green
mesh_terms: D001681:Biological Assay / Q000379:methods*; D020169:Caspases / Q000378:metabolism*; D005470:Fluorometry; D006801:Humans; D066298:In Vitro Techniques; D013379:Substrate Specificity
chemical_list: D020169:Caspases