Detail of "158584-09-9"

Reference

Evaluation of an imaging platform during the development of a FRET protease assay

Evaluation of an imaging platform during the development of a FRET protease assay. George, Jeanette; Teear, Michelle L.; Norey, Christopher G.; Burns, D. Dougal (Amersham Biosciences UK Limited, Buckinghamshire, UK). Journal of Biomolecular Screening, 8(1), 72-80 (English) 2003 Sage Publications. CODEN: JBISF3. ISSN: 1087-0571. DOCUMENT TYPE: Journal CA Section: 9 (Biochemical Methods) Synthetic peptide substrates labeled with a fluorescent donor and quenching moiety flanking an enzyme cleavage site provide a reliable method for monitoring enzyme activity. The dye pair Mca/Dnp has been widely used for this purpose, but poor soly. characteristics, combined with fluorescence emission in the region of the spectrum assocd. with interference from biologicals and library compds., can limit the usefulness of Mca/Dnp substrates in a high-throughput screening (HTS) environment. Peptide Mca-Arg-Pro-Lys-Pro-Val-Glu-Nva-Trp-Arg-Lys(Dnp)-NH2 is a matrix-metalloproteinase 3 (MMP-3) enzyme substrate that the authors have labeled with a CyDye pair, Cy3/Cy5Q. The Mca/Dnp- and CyDye-labeled substrates were compared during the development of an MMP-3 inhibitor assay. The results obtained showed that although the peptide substrates behaved similarly throughout the development of the MMP-3 assay, during a test screen of 934 compds. randomly selected from a collection of more than 70,000 compds., the CyDye substrate was considerably more reliable. Screen Z factor values of 0. 158584-09-9 and 79955-99-0 are also in the experiment.84 and 0.15 were obtained using the CyDye and Mca/Dnp peptides resp., and the authors found that although < 1% of the test compds. were auto-fluorescent at Cy3 wavelengths, > 10% could not be screened using the Mca/Dnp substrate because of compd. auto-fluorescence and interference. During this study, the authors used a PMT-based fluorescence plate reader and at the same time evaluated a charged couple device (CCD)-based imaging platform specifically optimized for use with CyDye reagents. The imaging platform gave improved read accuracy and faster plate processing times compared with the PMT reader. Overall, the results presented here highlight the potential benefit of employing the red-shifted CyDye reagents and imaging technol. during the development and execution of HTS protease screens. .