| Assay Method Information | |
| | ELISA-Based p53-MDM2 Binding Assay |
| Description: | ELISA plates were coated with equivalent amounts of either glutathione S-transferase (GST) protein or GST-MDM2 (1-188) fusion protein and incubated on a microtiter plate shaker overnight at 4 deg C. After washing the wells five times with PBS containing 0.2 M NaCl, the plates were incubated at 37 deg C with blocking solution (PBS containing 5% nonfat dry milk) for 2 hours. After the plates were washed five times, the GST-p53 was added, and the plates were incubated for an additional 30 minutes at 37 deg C. After another washing step, the plates were incubated with the anti-p53 monoclonal pAb421 (Oncogene Science, Inc.) in blocking solution for 1 hour at 37 deg C. The plates were again washed five times and incubated for 1 hour at 37 deg C with a goat anti-mouse IgG alkaline phosphatase conjugate (Promega, Inc.). Excess antibody was removed with 15 washes, the wells were filled with PBS, and the absorbance at 405 nm was read to establish background readings for the wells. After aspiration of the PBS, coupled antibody was incubated with p-nitrophenyl phosphate reagent solution (Sigma-Aldrich, Inc.). Finally, the A405 was measured with a microplate spectrophotometer (Molecular Devices, Inc.). Data analysis was performed using Microsoft Excel 98 (Microsoft Corp.) and DeltaGraph Pro (DeltaPoint, Inc.). Absorbance values were normalized by subtraction of equivalent GST-seeded well values from their GST-mdm2 (1-188)-seeded neighbors. IC50 values were determined by fitting data to a three-parameter sigmoidal dose-response function. |
| Affinity data for this assay | |
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