Activation of ICE-family proteases/caspases initiates apoptosis in mammalian cells. The CPP32/Caspase-3 Fluorometric Protease Assay provides a simple and convenient means for assaying the Ac-DEVD-dependent caspase activity. The assay is based on detection of cleavage of substrate Ac-DEVD-AFC. Ac-DEVD-AFC emits blue light (λmax = 400 nm); upon cleavage of the substrate by CPP32 or related caspases, free AFC emits a yellow-green fluorescence (lmax = 505 nm), which can be quantified using a fluorometer or a fluorecence microtiter plate reader. Z-DEVD-FMK (668 Daltons) is an irreversible and cell permeable inhibitor of caspase-3. This inhibitor can be used to inhibit caspase-3 activity and to study events downsteam of caspase-3 activation.
Store undiluted at -20°C. Reconstitute the substrate before use. Reconstitute in 1 ml DMSO to yield 1 mg/ml peptide in DMSO. Store the reconstituted substrate at –20°C for up to 1-2 months and avoid repeated freeze-thaw cycles.
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