Caspase-3 DEVD-R110 Fluorometric and Colorimetric Assay Kit provides a simple assay system for fast and highly sensitive detection of caspase-3 activity either by fluorescence or absorbance in mammalian cells. Biotium also offers the Caspase-3 DEVD-R110 Fluorometric HTS Assay Kit (30009), a one-step fluorescent assay for high throughput screening (HTS). For real-time detection of caspase-3 activity in intact cells, learn about our novel NucView® Caspase-3 Substrates.
- Caspase-3 activity measured with substrate (Ac-DEVD)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-DEVD)2-R110
- Enzyme inhibitor Ac-DEVD-CHO
About this kit
Caspase-3 is an active cell-death protease involved in the execution phase of apoptosis. The fluorogenic and chromogenic substrate (Ac-DEVD)2-R110 contains two DEVD tetrapeptides and is completely hydrolyzed by the caspase-3 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 (rhodamine 110) (Ex/Em= 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.
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-DEVD-CHO, which is a caspase-3 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-3 activity.
Note: While caspase-3 preferentially cleaves the consensus sequence DEVD compared to other substrate sequences, other caspases can also cleave DEVD efficiently. Overlapping caspase substrate recognition limits the usefulness of caspase substrate peptides for distinguishing between different caspase activities in cell lysates.
NucView® enzyme substrate technology is covered by U.S. patents.