| Assay Method Information | |
| | AlphaScreen Assay |
| Description: | When two proteins interact (one of which is conjugated to the donor bead and the other to the acceptor bead) the beads are brought into close proximity. Excitation of a photosensitizer in the donor bead results in the production of singlet oxygen that diffuses to and reacts with a chemiluminescent molecule in the acceptor bead. This results in the activation of fluorophores in the acceptor bead which results emitted light that can be detected. In this assay, GST-MDM2 (1-150) and full-length His6-wt-p53 were expressed in E. coli and affinity purified under non-denaturing conditions. To detect the MDM2-p53 interaction by the AlphaScreen assay (Perkin Elmer), GST-MDM2, His6-p53, and potential inhibitors were mixed in binding buffer, and incubated for 1 h at 24 deg C. Nickel acceptor beads and glutathione donor beads were added. Following 1 h incubation at 24 deg C, the mixture was analyzed in a fluorometer at an excitation wavelength of 680 nm. Nutlin-3a obtained from Cayman Chemical was used as a control. The IC50 values reported are from runs repeated four times. |
| Affinity data for this assay | |
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