| Assay Method Information | |
| | MDM2-p53 TR-FRET Binding Assa |
| Description: | Test compounds (12.34 uM stock in DMSO) were diluted three fold in series in DMSO and 2 ul per well were added into 384-well polypropylene plates (Matrix) in triplicates. GST-tag full length MDM2 (22 nM, 30.8 ul/well) in Assay Buffer (50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 1 mM DTT and 0.2 mg/ml BSA) were added followed by 5 ul per well of 72 nM biotin p53 peptide (Biotin-Aca-SQETFSDLWKLLPEN-OH) in Assay Buffer. The samples were incubated at room temperature for 30 min, and 2.5 ul per well of detection solution containing 16 nM europium (Eu) conjugated streptavidin (PerkinElmer) and 200 nM allophycocyanin (APC) conjugated anti-GST antibody (Columbia Biosciences) in Assay Buffer (without DTT) were added. The samples were incubated at room temperature for 40 min. Assay signals were monitored by reading excitation at 340 nm and emission fluorescence at 615 nm and 665 nm on an Envision reader. IC50 values were calculated using Prism software (GraphPad). |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |