| Assay Method Information | |
| | p53-MDM2 Binding ELISA |
| Description: | Interference of the p53-MDM2 binding by test compounds was measured in a 96-well polypropylene round-bottom microtiter plate (Costar, Serocluster). Human MDM2 (amino acids 1-118) was preincubated with 10% DMSO or compounds in assay buffer. After 15-min incubation of the sample, a biotinylated p53-derived peptide was added. As a negative control, buffer only was added into separate wells (blanks). After another 30 min, the incubation mixture was added to 96-well plates coated with streptavidin. After 1 h, the wells were extensively washed. Then, the MDM2 specific antibody was added. After 1 h, the wells were thoroughly washed and the secondary antibody (anti-rabbit-IgG-POD) was added. After another hour, the wells were washed, and the peroxidase subtrate ABTS was added. After 30 min, OD 405/490 nm was determined with a Dynatech MR 7000 ELISA reader. Compounds were titrated to determine IC50 values twice (range of compound concentration: 0.5, 1, 5, 10, 25, 50, 125, 250 uM). |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |