Caspase assays: identifying caspase activity and substrates in vitro and in vivo

The measurement of general caspase activity and the quantification of purified recombinant caspases in vitro can be accomplished with relative ease. But the determination of which caspases are active in a cellular context is much more challenging. This is because commercially available small molecule substrates and inhibitors do not display sufficient specificity to dissect the complex interplay of caspase pathways. Here we describe procedures that can be used to validate which caspases are active in cell culture models and determine which caspases are responsible for specific cleavage events. We also recommend methods for working with recombinant initiator caspases in vitro and suggest ways to accurately assess the cleavage efficiency of natural caspase substrates.