2.5. MDM2 binding assay

MDM2 protein generation and binding studies were performed as described. ¹⁷ Briefly, binding was performed using MDM2 (1–125) protein titrated against 50 nM carboxyfluorescein (FAM)—labeled 12/1 peptide13 (FAM‐RFMDYWEGL‐NH2). The apparent K _D value of FAM‐labeled 12/1peptide was determined to be 13.0 nM and subsequently used to determine the apparent K _D values of the respective competing ligands in competition assays in fluorescence anisotropy experiments. Titrations were carried out with the concentration of MDM2 held constant at 250 nM and the labeled peptide at 50 nM. The competing molecules were then titrated against the complex of the FAM‐labeled peptide and protein. Curve‐fitting was carried out using Prism 4.0 (GraphPad) to determine K _D values. Readings were carried out with an Envision Multilabel Reader (PerkinElmer). Experiments were carried out in PBS (2.7 mM KCl, 137 mM NaCl, 10 mM Na₂HPO4, and 2 mM KH₂PO₄ [pH 7.4]) and 0.1% Tween 20 buffer. All titrations were carried out in triplicate.