Assay Method Information

Assay Name:  Tryptophan Fluorescence Quenching Assay (Kd)
Description:  Binding was also measured by a competition assay with a noncovalently bound inhibitor (Kd=4.6 uM) that quenched tryptophan fluorescence upon binding in the protease active site. Then 3.5 uM protein was titrated by this competitive inhibitor (from 0 to 40 uM) in the absence or presence of 50 uM test compound. An aliquot of each dilution was transferred to a UV-star Greiner 96-well microplate. After 1 h of incubation at room temperature, fluorescence was measured at 25 deg C on a Biotek Synergy4 microplate reader with exe=280 nm (bandwidth 10 nm) and em=340 nm (bandwidth 20 nm). Fluorescence intensities were corrected for inner-filter effect. Kd values of compounds were inferred from their effect on the Kd of the reference compound.
Affinity data for this assay

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