Development of AlphaScreen assay.

<p>(A) AlphaScreen assay to detect binding between p53 and MDM2. Crude translation mixtures of biotinylated p53 (WT or ∆I) and FLAG-MDM2 (0.75 μL each) were used for the assay. FLAG-DHFR was used as a control for MDM2. (B) A reaction mixture containing p53 and MDM2 prepared under the same conditions as those in (A) was mixed with 2, 10, or 50 μM Nutlin-3. Biotinylated FLAG-peptide was used as a control to measure the interference of Nutlin-3 in the AlphaScreen assay. (C) Validation of the quality of the AlphaScreen assay. Z′ factor was calculated from the reaction of wild-type p53 and MDM2 (positive control, n = 40, square) and the reaction of p53 ∆I mutant and MDM2 (negative control, n = 40, circle). In (A) and (B), all data are expressed as mean values of three independent experiments with error bars indicating standard deviations.