Caspase-8 Fluorometric and Colorimetric Assay Kit provides a simple assay system for fast and highly sensitive detection of caspase-8 activity either by fluorescence or absorbance in mammalian cells. Biotium also offers the Caspase- IETD-R110 Fluorometric HTS Assay Kit (30012), a one-step fluorescent assay for high throughput screening (HTS)
- Caspase-8 activity measured with substrate (Ac-IETD)2-R110
- Compatible with both fluorometric and colorimetric detection systems
- Fast enzyme kinetics
- Enzymatic reaction forms yellow colored and green fluorescent R110 product
- Endpoint assay requires cell lysis
- Cell lysis buffer
- Assay buffer
- Enzyme substrate (Ac-IETD)2-R110
- Enzyme inhibitor Ac-IETD-CHO
About this kit
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. The fluorogenic and chromogenic substrate (Ac-IETD)2-R110 contains two IETD tetrapeptides and is completely hydrolyzed by the caspase-8 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 (rhodamine 110) (Ex/Em= 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.
This assay kit includes Ac-IETD-CHO, which is a caspase-8 inhibitor and can be used as a negative control. R110 is also 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.