| Assay Method Information | |
| | Time Resolved-Fluorescence Resonance Energy Transfer (TR-FRET) Assay |
| Description: | Test compounds in dimethyl sulfoxide were acoustically transferred into a white 384-well low volume assay plate (Perkin Elmer #6008289) using a Labcyte Echo and Access robot, employing a 3-fold dilution strategy across 11 points starting at 1250 μM. 8 mL of a protein (Bcl-2 or Bcl-xL)/probe/antibody mixture pre-equilibrated for one hour in assay buffer was added to each well at final concentrations. The samples were then mixed on a shaker for 1 minute and equilibrated for an additional 3 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 (Perkin Elmer) using a 340/35 nm excitation filter and 520/525 (F-Bak) and 495/510 nm (Tb-labeled anti-GST antibody) emission filters. Inhibition constants (Ki) were determined using Wang's equation |
| Affinity data for this assay | |
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