Caspase-3 DEVD-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-3 activity by fluorescence in enzymatic reaction or mammalian cells. The fluorogenic substrate (Ac-DEVD)2-R110 contains two DEVD tetrapeptides and is completely hydrolyzed 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 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.
Note: While caspase-3 preferentially cleaves the consensus sequence DEVD compared to other substrate sequences, other caspases also can cleave DEVD efficiently. Overlapping caspase substrate recognition limits the usefulness of caspase substrate peptides for distinguishing between different caspase activities in cell lysates.
Kit Components:
- Cell lysis/assay buffer
- Enzyme substrate (Ac-DEVD)2-R110
- Enzyme inhibitor Ac-DEVD-CHO
- R110
Features:
- HTS-compatible: Single-step homogenous assay specifically designed for HTS-based detection.
- Fast: Fast enzyme kinetics.
- Sensitive: Green fluorescent readout
For real-time detection of caspase-3 activity in intact cells, see our novel NucView™488 Caspase-3 Substrate.
References
1. Cell Death Diff. 6, 99 (1999).
2. J Biol Chem 274, 11549 (1999).
3. J Biol Chem 275, 288 (2000).
4. Biochemistry, 38, 13906 (1999).
