| Assay Method Information | |
| | Competitive FP Binding Assay |
| Description: | The Fluorescence Polarization (FP) competitive binding assays were performed to accurately determine the binding affinities of our DCN1 inhibitors. A novel FAM labeled fluorescent probe compound (46) was designed and synthesized based on one of our potent small molecule DCN1 inhibitors. Equilibrium dissociation constants (Kd) values of 46 to both DCN1 and DCN2 proteins were determined from protein saturation experiments by monitoring the total FP values of mixtures composed with the fluorescent probe at a fixed concentration and proteins with increasing concentrations up to full saturation. Serial dilutions of proteins were mixed with 46 to a final volume of 200 μl in the assay buffer (100 mM phosphate buffer, pH=6.5, with 0.02% Tween-20 and 2% DMSO). Final probe concentration was 5 nM for both assays. Plates were incubated at room temperature for 30 minutes with gentle shaking to assure equilibrium. FP values in millipolarization units (mP) were measured using the Infinite M-1000 plate reader (Tecan U.S., Research Triangle Park, NC) in Microfluor 1 96-well, black, round-bottom plates (Thermo Scientific, Waltham, MA) at an excitation wavelength of 485 nm and an emission wavelength of 530 nm. Kd values of 46 were calculated by fitting the sigmoidal dose-dependent FP increases as a function of protein concentrations using Graphpad Prism 6.0 software (Graphpad Software, San Diego, CA). |
| Affinity data for this assay | |
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