Assay Method Information

Assay Name:  Fluorescent Peptide Displacement Assay
Description:  Test compound was incubated for 15 minutes with 30 nM peptide fluorescein-FMDYWEGLN (Fl 9mer) and 120 nM (glycine-HDM2 17-125) in assay buffer. The polarization of the fluorescein label was measured by excitation at 485 nm and emission at 530 nm. Percent inhibition was calculated from the decrease in the fluorescence polarization of the labeled peptide upon displacement from HDM2 by the compounds, using buffer with Fl 9mer but without HDM2 as background. Data were analyzed by non-linear least squares fitting to a hyperbolic binding function. The measured IC50s of the p53 wild-type (QETFSDLWKLLP) and optimized (MPRFMDYWEGLN) peptides are 2000 and 250 nM, respectively. Under these conditions, the IC50 is 2.5 fold higher than the dissociation binding constant (Kd).
Affinity data for this assay
 

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