| Assay Method Information | |
| | Fluorescence Polarization Competition Assay |
| Description: | The fluorescence polarization assays were performed in 96 well polypropylene F-bottom black microplates (Greiner Bio-One) with a final volume of 100 μL. During the competition assay, a fluorescently-labeled Bak-BH3 peptide (FITC-Ahx-GQVGRQLAIIGDDINR-CONH2, hereafter FITC-Bak , where FITC=fluorescein isocyanate; Ahx=6-aminohexanoyl linker) was competed off of either MCL-1172-327, BCL-XL 1-212 or BCL-21-211 with the synthesized inhibitors. The binding affinities of FITC-Bak to MCL-1, BCL-XL and BCL-2 were determined via a fluorescence polarization assay where various concentrations of the selected proteins were titrated into solutions of 10 nM FITC-Bak in 20 mM HEPES, pH 6.8, 50 mM NaCl, 3 mM DTT, 0.01% Triton X-100 and 5% DMSO at room temperature. The changes in the fluorescence polarization were then measured using a BMG PHERAstar FS multimode microplate reader equipped with two PMTs for simultaneous measurements of both the perpendicular and parallel fluorescence emission at a 485 nm excitation and 520 nm emission filter. Regression analysis was then performed on the polarization data using Origin (OriginLab, Northampton, Mass.) and the data was fitted to the Hill equation, thus producing binding curves for FITC-Bak with MCL-1, BCL-XL and BCL-2. |
| Affinity data for this assay | |
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