| Assay Method Information | |
| | MDM2-p53 Interaction Using a 96-Well Plate Binding Assay (ELISA) |
| Description: | For initial testing, the compounds and controls were plated out in triplicate into clear 96-well plates (Nunc) in 10 ul aliquots to give final concentrations of 500 uM, 100 uM, and 20 uM in the assay. Control samples consisted of 5% DMSO carrier alone as a negative control and 100 nM active peptide (AP-B: Ac-Phe-Met-Aib-Pmp-6-Cl-Trp-Glu-Ac3-Leu-NH2) as a positive control peptide antagonist of the MDM2-p53 interaction (IC50 = 5 nM). Compounds and controls aliquoted in 96-well plates were preincubated with in vitro translated MDM2, before transfer of the MDM2-compound mixture to the b-IP3 streptavidin plates, and incubation at 4 deg C for 90 min. After washing with PBS and subsequently incubating with primary mouse monoclonal anti-MDM2 antibody and goat-anti-mouse horseradish peroxidase (HRP) conjugated secondary antibody. The bound HRP activity was measured by enhanced chemiluminescence (ECL, Amersham Biosciences) using the oxidation of the diacylhydrazide substrate, luminol, to generate a quantifiable light signal. The luminol substrate together with enhancer was automatically injected into each well and the relative luminescence units (RLU) measured over a 30 s interval using a Berthold MicroLumat-Plus LB 96 V microplate luminometer. The IC50 was calculated using a plot of % MDM2 inhibition versus concentration and is the average of three independent experiments. |
| Affinity data for this assay | |
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