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| | Fluorescence Polarization-Based (FP-Based) Binding Assay |
| Description: | The dose-dependent binding experiments were carried out with serial dilutions of the tested compounds in DMSO. A 5 ul sample of the tested samples and preincubated MDM2 protein and PMDM6-F peptide were added in Dynex 96-well, black, round-bottom plates (Fisher Scientific). For each assay, the controls included the MDM2 protein and PMDM6-F (equivalent to 0% inhibition), only PMDM6-F peptide (equivalent to 100% inhibition). The polarization values were measured after 3 hrs of incubation using an ULTRA READER (Tecan U.S. Inc., Research Triangle Park, NC). The IC50 values, i.e. the inhibitor concentration at which 50% of bound peptide is displaced, were determined from a plot using nonlinear least-squares analysis. Curve fitting was performed using GRAPHPAD PRISM software (GraphPad Software, Inc., San Diego, CA). To calculate the binding affinity constants (Ki) of inhibitors, a web-based computer program was developed for computing the Ki values for inhibitors in FP-based binding assays based upon an equation. |
| Affinity data for this assay | |
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