Caspase-8 IETD-R110 Fluorometric HTS Assay Kit is specifically designed for HTS-based assays. The kit provides a homogenous assay system for fast and highly sensitive detection of caspase-8 activity by fluorescence in enzymatic reaction or mammalian cells. The fluorogenic substrate (Ac-IETD)2-R110 contains two IETD tetrapeptides and is completely hydrolyzed 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.
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.
The 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.
- Cell lysis/assay buffer
- Enzyme substrate (Ac-IETD)2-R110
- Enzyme inhibitor Ac-IETD-CHO
- HTS-compatible: Single-step homogenous assay specifically designed for HTS-based detection.
- Fast: Fast enzyme kinetics.
- Sensitive: The enzymatic reaction forms intensely green fluorescent rhodamine 110 (R110) product.
1) Biochemistry 38, 13906 (1999).