Fluorescence Polarization Screening Assays for Small Molecule Allosteric Modulators of ABL Kinase Function
A) To characterize the baseline FP signal, the 6-carboxy-fluorescein labeled probe peptides p41 (red), p40 (green), p8 (blue), and 3BP-1 (black) were serially diluted in the concentration range of 1–1000 nM. The FP signals (solid lines, left Y axis) and corresponding fluorescence intensities (dashed lines, right Y axis) were measured and plotted as a function of peptide concentration. Average values are shown ± SE from four measurements per condition. B) To test for probe peptide interaction with ABL N32L by FP, each peptide (50 nM) was incubated with the ABL N32L protein over the range of 0.08–25 μg/well. The resulting FP signals were corrected for baseline FP signal recorded in the absence of the N32L protein and plotted against the N32L protein concentration. Average FP values are shown ± SE from four measurements per condition; error bars are smaller than the diameter of some data points.