Optimization of FRET Assay with the Varioskan® Spectral Scanning
Microtiter Plate Fluorometer
Arja Lamberg, Vuokko KytOniemi, Hanna GranO and Jorma Lampinen, Thermo Fisher Scientific, Vantaa, Finland
Introduction
This paper presents the principle of optimization for fluorometric assays. Optimization is based on the measurement of fluorescence spectra with the Thermo Scientific Varioskan microtiter plate spectral scanning fluorometer. The optimal assay settings for the excitation and emission wavelengths are determined from this spectral data. The optimization procedure is shown with the FRET technology based QuantiCleaveTM protease assay from Pierce Biotechnology Inc, Rockford, IL (product number 23267) and the ApoTarget Caspase-3 assay (product number KHZOO12) from BioSource International Inc, Camarillo, CA.
FRET (Fluorescence Resonance Energy Transfer) is an emerging fluorometric assay technology commonly used in proteomics. It is based on the use of two fluorometric labels, one acting as a donor and another as an acceptor molecule. When these molecules are in close proximity, emission energy of the donor is used for the excitation of the acceptor. With the long distance between the molecules the excitation of the acceptor is not possible. A common variation of this structure is to use one fluorometric label and one quencher that completely quenches the emission when in close proximity to the fluorescent label. In this case the fluorescent signal is detected when the quencher is removed.
QuantiCleave Fluorescent Protease Assay is based on a fluorescein-casein conjugate. The fluorescein label on the FTC-casein conjugate is highly quenched due to FRET based fluorescence homotransfer. The fluorescence which is observed in the homotransfer FRET mode occurs by way of electronic energy transfer between identical fluorophores. In this system, fluorescein acts both as the energy donor and acceptor. Protease activity is evidenced by an increase in fluorescence as the FTC-casein conjugate is digested by the protease. Fluorescence quenching is relieved and an increase in fluorescence is observed.
ApoTarget Caspase-3 protease assay is based on the recognition of the DEVD (Asp-Glue-Val-Asp) amino acid sequence linked to the fluorometric label AFC (7-amino-4-trifluoromethyl coumarin) by Caspase-3. The reaction substrate, DEVD-AMC, does not show the typical fluorometric emission with the linked peptide, but the fluorescence is restored when AFC is cleaved off the DEVD peptide. The resulting fluorescence is quantified and is proportional to Caspase-3 activity in the sample.