Fluorescence Polarization Screening Assays for Small Molecule Allosteric Modulators of ABL Kinase Function
A) The p41 FP probe binds the ABL N32L protein through the SH3 domain. The p41 probe peptide (50 nM) was combined with wild type, HAL, and W118A ABL N32L proteins over the range of concentrations shown. The resulting FP signals were measured and plotted as a function of N32L protein concentration. B) FP assay stability. The p41 probe peptide (50 nM) was combined with the three ABL N32L proteins (12.8 μg/well) and FP signals were recorded over the time course shown. C) DMSO tolerance. FP assays consisting of the p41 probe peptide (50 nM) and each ABL N32L protein (25 μg/well) were incubated with the DMSO concentrations shown, and FP signals were recorded 1 h later. D) Unlabeled peptide competition. For the competition assay, the p41 probe peptide (50 nM) was mixed with unlabeled p41 peptide or a negative control peptide of unrelated sequence (QKEGERALPSIP) and similar length (Con) over the range of concentrations shown. The ABL N32L protein (20 μg/well) was then added, and FP signals were recorded. FP signals were corrected for the background p41 peptide FP signal and plotted as a function of the unlabeled peptide concentration. In all experiments (A through D), average FP values are shown ± SE from four measurements per condition.