| Assay Method Information | |
| | TR-FRET Binding A |
| Description: | Compound IC50 values were assessed following a 10-point, half-log10 dilution schema starting at 10 uM compound concentration. The assay was performed in 384-well LV plates (Matrix 4365, Thermo Scientific) and run in the presence and absence of the compound of interest. Each well of 12 uL assay mixture contained 10 mM Tris (pH 7.4), 1 mM DTT, 0.005% Tween-20, 150 mM NaCl, 10% DMSO, 3 nM GST-Mcl-1, 1 nM LanthaScreen Eu-tagged GST antibody (#PV5594, Invitrogen, Life Technologies), and 8 nM fluorescently labeled peptide. Reactions were incubated at 24 °C for 60 min before reading on a Tecan F500 spectrofluorometer with excitation at 340 nm and emission at 620 nm and 665 nm. Subsequently, the ratio of fluorescence emission intensity at 665 nm to 620 nm was calculated for each reaction, and the dose response of the ratio to testing compound concentration was fitted to a select fit model that will provide the best fit quality using automatic parameters to derive IC50 values for each testing compound. |
| Affinity data for this assay | |
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