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Caspase-3/7 Apoptosis Assay Kit provides a method for detecting apoptosis by providing a simple and reliable method for assaying caspase-3/7 activity. The kit can be used to continuously measure the activity of caspase-3/7 in cell extracts and purified enzyme preparations, using a fluorometer or fluorescence microplate reader. The rhodamine 110-derived substrate (Z-DEVD-R110) used in this assay is a non-fluorescent bisamide compound that, upon enzymatic cleavage, is converted in a two-step process to the fluorescent monoamide and then to the even more fluorescent R110 product. Both of these hydrolysis products exhibit spectral properties similar to those of fluorescein, with peak excitation and emission wavelengths of 496 nm and 520 nm, respectively. In addition to the Z-DEVD–R110 substrate, the Caspase-3/7 Z-DEVD-R110 Assay Kit contains the reversible aldehyde inhibitor Ac-DEVD-CHO, as well as the reference standard R110. The Ac-DEVD-CHO inhibitor confirms that the fluorescence signals in both induced and control cell populations are due to the activity of caspase-3/7. The reference standard allows for quantification of the amount of R110 released in the reaction.
Specifications
1. Platform: Microplate Reader, Fluorometer
2. Detection Method: Fluorescent
3. Ex/Em: 496/520 nm
Applications
Caspase Activity Assay
Components
1. Cell Lysis Buffer: 10 ml
2. Cell Assay Buffer: 10 ml
3. Enzyme Substrate Z-DEVD-R110: 250 µl
4. Enzyme Inhibitor Ac-DEVD-CHO: 20 µl
5. Standard R110: 1 ml
Storage
Store at -20°C and protect from light.
Case Study
Detection of apoptosis activity in Jurkat cells using the Assay Kit. Cells were either treated with 10 μM camptothecin for four hours at 37°C to induce apoptosis (induced) or left untreated(control). Both induced and control cells were then harvested, lysed and assayed as described in the kit protocol. Reactions were carried out at room temperature and fluorescence was measured in a fluorescence microplate reader using excitation at 470 ± 10 nm and emission detection at 520 ± 10 nm after the indicated amount of time
