Caspase-8 is the most upstream caspase in the CD95/Fas apoptotic pathway and is activated by the signaling pathways for CD95/Fas and TNF. Caspase-8 Fluorometric and Colorimetric Assay Kit provides a simple assay system for fast and sensitive detection of caspase-8 activity either by fluorescence or absorbance. The kit includes the fluorogenic substrate (Ac-IETD)2-R110, the enzyme inhibitor Ac-IETD-CHO as a negative control, R110 as a standard and optimized lysis and assay buffers. The substrate(Ac-IETD)2-R110 contains two IETD tetrapeptides and is completely hydrolyzed by the enzyme in a two-step process. Cleavage of the first IETD 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.
Although fluorometric detection of the end products is preferred because of the superior sensitivity, detection by absorbance is also possible. In fact, the extinction coefficient of R110 is 10 times higher than that of p-nitroaniline (pNA), a dye commonly used in chromogenic substrates, making R110-based substrates significantly more sensitive than pNA-based substrates, even by colorimetric detection.
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.
- Cell lysis buffer
- Assay buffer
- Enzyme substrate (Ac-IETD)2-R110
- Enzyme inhibitor Ac-IETD-CHO
- Fast: Fast enzyme kinetics.
- Sensitive: The enzymatic reaction forms intensely yellow colored and highly green fluorescent rhodamine 110 (R110) product.
- Versatile: Compatible with both fluorometric and colorimetric detection systems.
1) Biochemistry 38, 13906 (1999).