Fluorescence Polarization Assays for Protein-Ligand Interactions

Direct and competitive FP assays were performed as described^{13 (link)}. Direct binding curves were generated by incubating serial dilutions of protein with FITC–BID BH3 (15-60 nM) in 96-well format and plates read at equilibrium using a SpectraMax M5 microplate reader (Molecular Devices). For competitive FP assays, a serial dilution of small molecule was incubated with the indicated concentration of protein for 20 minutes at room temperature. FITC–BID BH3 was then added to the wells at the indicated concentration and the plate read at equilibrium. For dilution assays, MCL-1ΔNΔC was reduced using TCEP resin (Pierce) for 1 hour according to the manufacturer's protocol, followed by the addition of MAIM1 at 5x molar excess, unless otherwise indicated. Conjugation reactions were performed at room temperature for 45 minutes followed by removal of excess small molecule by spin column (Biorad) or overnight dialysis into FPLC buffer (150 mM NaCl, 50 mM Tris, pH 7.4). Protein concentration was determined by NanoDrop (Thermo) and then applied in FP assays, performed as described above. Data were analyzed by nonlinear regression analysis using Prism software (GraphPad).