| Assay Method Information | |
| | Caspase-3 Inhibition Assay |
| Description: | Caspase-3 was assayed at 23 deg C (room temp) in 96-well plates using the internally quenched tetrapeptide substrate. Enzymatic cleavage between the aspartate and the AFC fluorophore liberates 7-amino-4-trifluoromethyl coumarin which is detected using an excitation wavelength of 400 nm and an emission wavelength of 505 nm in a SpectraMax GeminiXS plate reader operated at room temperature. A steady state rate of substrate cleavage is obtained for analysis. For IC50 determination, typically 11 concentrations were freshly prepared by serial dilution with assay buffer containing no cysteine with substrate added to the assay well. Once substrate and inhibitor were added to the assay plate, the reaction was initiated by addition of enzyme, prepared in assay buffer containing cysteine. AFC production was monitored continuously for 90 min by exciting at 400 nm and measuring the emission at 505 nm every 42 s. |
| Affinity data for this assay | |
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