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Assay Method Information

Assay Name:  Time Resolved-Fluorescence Resonance Energy Transfer (TR-FRET) Assay
Description:  The measurement of competition of compounds of Formula (I) with F-Bak for a Bcl-2 family protein (Bcl-xL) binding site using a Time Resolved Fluorescence Resonance Energy Transfer (TR-FRET) binding assay:Test compounds were serially diluted in DMSO starting at 50 .mu.M (2.times. starting concentration; 10% DMSO) and 10 .mu.L transferred into a 384-well plate. The samples are then mixed on a shaker for 1 minute then incubated for an additional 2 hours at room temperature. For each assay plate, a probe/antibody and protein/antibody/probe mixture were included as a negative and a positive control, respectively. Fluorescence was measured on the ENVISION plate reader (Perkin Elmer) using a 340/35 nm excitation filter and 520/525 (F-Bak) and 495/510 nm (Tb-labeled anti-his antibody) emission filters. Dissociation constants (K.sub.i) were determined using Wang's equation (Wang, Z. X.).
Affinity data for this assay
 

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Last update November 1, 2007
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