Optimization of fluorometric Caspase-3 assay with Varioskan® spectral scanning

Abstract

In this paper, we present the principle of the assay optimization for fluorometric Caspase-3 assays. It is based on the measurement of fluorescence spectra with the Thermo Varioskan spectral scanning fluorometer. From this spectral data, the optimal assay settings for the maximum sensitivity are determined, including excitation and emission wavelengths. The optimization procedure is shown with ApoTarget™ Caspase-3 assay (product number KHZ0012) from BioSource International Inc, Camarillo, CA.
Caspase-3 belongs to Interleukin-1ß Converting Enzyme (ICE) family of cysteine proteases and is present in cells as an inactive 32 kDa proenzyme. It is activated specifically during apoptosis and is therefore one of the most important markers for apoptotic activity. ApoTarget Caspase-3 protease assay is based on the recognition of the DEVD (Asp-Glue-Val-Asp) amino acid sequence linked to fluorometric label AFC (7-amino-4-trifluoromethyl coumarin) by Caspase-3. Free AFC has a fluorometric emission maximum at 505 nm but when AFC is linked in the DEVD peptide, this emission is quenched by FRET (fluorescence resonance energy transfer). Fluorescent emission maximum at 505 nm is restored when AFC is cleaved off the DEVD peptide. The resulting fluorescence is quantitated and is proportional to Caspase-3 activity in the sample.