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Related Links Caspase Cleavage and Activation Physiological Caspase Substrates and Inhibitors Mitochondrial Changes in Apoptosis |
One approach to directly assess the activity of purified caspases or caspases enriched from cell extracts involves the use of peptide substrates conjugated to a colorimetric or a fluorescent reporter molecule, as shown below. When caspases cleave these peptide substrates, the reporter molecule is released and measured by a spectrophotometer or fluorometer, lending them to multi-well microplate based assays. These caspase assays provide a quantitative analysis of caspase activation because the colorimetric or fluorometric signal is proportional to the enzymatic activity, and serve as an effective platform for screening caspase inhibitors. 
Caspase-8 (catalog # NB705-C8) was preincubated with increasing amounts of pro-Caspase-3 (catalog # NB731-C3) for 1 hour. Caspase-3 activity (blue) was determined by measuring the cleavage of peptide substrate, DEVD-afc in a fluorescence plate reader using the Caspase-3 fluorometric assay kit. Background activity of pro-Caspase-3 (red) was measured by monitoring DEVD-afc cleavage in the absence of active caspase-8. Typical formats to monitor caspase activity in live cells by flow cytometry and microscopy use fluorescent labeled peptide inhibitors that irreversibly bind the catalytic site of select caspases. Peptide substrates and inhibitors provided in Novus’ caspase kits have been developed for optimal enzyme kinetics and high affinity binding. The amino acids in each synthetic peptide substrate, or inhibitor, represent the preferred consensus sequence recognized by individual caspases.
*Browse our available caspase activity kits/other products by clicking on your caspase of interest. Are there any limitations to using these kits?While detection of processed caspases as well as quantification of caspase activity have been widely used to examine the role of individual caspases, these peptides based tools have limited specificity. Data generated from cell lysates or live cells, containing multiple caspases, should be interpreted with caution. For instance, given the promiscuity and catalytic efficiency of Caspase-3, it will likely target additional peptides. Novus recommends performing complementary assays with siRNA/shRNA mediated gene silencing or using cells derived from caspase knockout animals to corroborate the contribution of individual caspases. Learn About Caspase Cleavage/Activation |
