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Discovery Technologies, Roche, 340 Kingsland St., Nutley, NJ, 07110
Proteases are an important target family for drug development due to their critical roles in protein activation, cell regulation and signaling. FRET assay is commonly used to detect protease activity. However, compound interference, which leads to high false positive or false negative rate in HTS campaign, is a concern for this assay format. To overcome this problem, we evaluated a new FRET-label pair developed recently by AAT Bioquest (Sunnyvale, California), which includes a time-resolved Tb-complex label and a non-fluorescent TideQuencherTM (TQ) dye quencher. The new peptide substrate, TQ2-XL-peptide-X2TRFFluo-Tb, gave very robust signal window using either the time-resoled fluorescent intensity (TRF) or fluorescence life time (FLT) readout. The time-resolved fluorescence feature of Tb-complex greatly decreased assay artifact caused by the short-lived autofluorescent compounds.