Caspase-3 and Caspase-8 Fluorometric HTS Assay Kits provide a homogenous assay system for fast and highly sensitive detection of Caspase-3 or Caspase-8 activity by fluorescence in enzymatic reaction or mammalian cells.

• Single-step homogenous assay specifically designed for HTS-based detection
• Fast enzyme kinetics
• Sensitive green fluorescent readout

Caspase-3 DEVD-R110 Fluorometric HTS Assay Kit includes the fluorogenic substrate (Ac-DEVD)2-R110, which contains two DEVD tetrapeptides and is completely hydrolysed by the enzyme in two successive steps. Cleavage of the first DEVD peptide results in the monopeptide Ac-DEVD-R110 intermediate, which has absorption and emission wavelengths similar to those of R110 (λEx/λEm = 496/520 nm) but has only about 10% of the fluorescence of the latter. Hydrolysis of the second DEVD peptide releases the dye R110, leading to a substantial fluorescence increase.

The Caspase-3 Fluorometric HTS Assay Kit includes Ac-DEVD-CHO, which is a caspase-3 inhibitor and can be used as a negative control. Also, R110 is provided in the kit for generating a standard curve, which can be used for quantifying caspase-3 activity. One mL cell lysis/assay buffer is sufficient for 10 assays in 96-well format.

Caspase-8 IETD-R110 Fluorometric HTS Assay Kit includes the fluorogenic substrate (Ac-IETD)2-R110, which contains two IETD tetrapeptides and is completely hydrolysed by the enzyme in two successive steps. Cleavage of the first IETD peptide results in the monopeptide Ac-IETD-R110 intermediate, which has absorption and emission wavelengths similar to those of R110 (λEx/λEm = 496/520 nm) but has only about 10% of the fluorescence of the latter. Hydrolysis of the second IETD peptide releases the dye R110, leading to a substantial fluorescence increase.

The Caspase-8 Fluorometric HTS Assay Kit includes Ac-IETD-CHO, which is a caspase-8 inhibitor and can be used as a negative control. Also, R110 is provided in the kit for generating a standard curve, which can be used for quantifying caspase-8 activity.

Note: While caspase-8 preferentially cleaves the consensus sequence IETD compared to other substrate sequences, other caspases such as caspase-3 also can cleave IETD efficiently. Overlapping caspase substrate recognition limits the usefulness of caspase substrate peptides for distinguishing between different caspase activities in cell lysates.