EnzChek™ Caspase-3 Activity Assay Kits

The EnzChek Caspase-3 Activity Assay Kit enables detection of 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.

Key features of the EnzChek Caspase-3 Activity Assay Kit includes:

• Fluorescent assay using standard fluorescein (FITC) excitation/emission settings
• Format allows for continuous measurement of caspase-3/7 activity in cell extracts
• Fluorescent and enzymatic controls included

The aminomethylcoumarin (AMC)-derived substrate (Z-aspartic acid-glutamic acid-valine-aspartic acid (DEVD)-AMC) that is included in the EnzChek Caspase-3 Activity Assay Kit is weakly fluorescent in the UV range (excitation/emission maxima ∼330/390 nm), but yields a bright, blue-fluorescent product (excitation/emission maxima ∼342/441 nm) upon proteolytic cleavage. The kit can be used to continuously measure the activity of caspase-3 and closely related proteases in cell extracts and purified enzyme preparations using a fluorometer or fluorescence microplate reader.

The rhodamine 110-derived substrate (Z-DEVD-R110) used in the EnzChek Caspase-3 Activity Assay Kit 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 substrates, the EnzChek Caspase Activity Assay Kit contains the reversible aldehyde inhibitor Ac-DEVD-CHO, as well as an AMC or R110 reference standard. The Ac-DEVD-CHO inhibitor confirms that the fluorescence signals in both induced and control cell populations are due to the activity of the caspase-3/7 protease. The reference standard allows for quantification of the amount of AMC or R110 released in the reaction.