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Fluorometric Protease Assays in the SpectraMax Gemini Microplate Spectrofluorometer: Example Using Caspase3 (MaxLine Application Note #35)

nd AFC appear to give identical sensitivity.3

Caspase3 is one of the family of cysteinecontaining proteases that cleave their substrates at an aspartate residue.4,5 It appears to have several natural substrates6, including poly(ADPribose) polymerase, in which case the cleavage site is at the tetrapeptide AspGluValAsp (DEVD).4,5 Although caspases are well known for their key roles in programmed cell death (apoptosis), an extracellular caspase3 like activity has been recently discovered in suspensions of live cells.7

Several coumarinlabelled DEVD substrates have been used to measure caspase3 activity. Typically AMC4 or AFC7,8 is attached to the Cterminus, and acetyl (Ac) or benzyloxycarbonyl (Z) is attached to the amino terminus. Enzymatic activity results in the release of free AMC (or AFC) product. There are several commercial sources of these substrates, as well as caspase3 assay kits containing them (including Molecular Probes, Enzyme Systems Products, Clontech, R&D Systems, GenoTechnology, CalBiochem, Peptides International, BioRad, and Pharmingen).

This application note describes an assay for caspase3 in the SPECTRAmax GEMINI microplate spectrofluorometer using the substrate ZDEVDAMC.


MATERIALS AND METHODS
ZDEVDAMC substrate was purchased as part of Molecular Probes EnzChek Caspase3 Assay Kit #1 (catalog #E13183). Recombinant caspase3 enzyme was purchased from Calbiochem (catalog #235417), and 7Amino4Methylcoumarin (AMC) from Sigma (catalog #A 9891). Black 96-stripwell plates and white-96 well plates were purchased from Corning Costar.

All dilutions of the substrate and enzyme were made with 1X Reaction Buffer, (prepared by diluting 200 L of the 5X Reaction Buffer and 5 L of DTT, both included in the kit, with 795 L of deionized water.) Stock ZDEVDAMC (10 mM) was prepared by adding 520 L of DMSO into the vial of
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