Active Mdm2 p53 Binding Protein Homolog (MDM2)

Mouse double minute 2 homolog (MDM2) also known as E3 ubiquitin-protein ligase. MdM2 is a cellular oncoprotein that recognizes the N-terminal trans-activation domain (TAD) of the p53 tumor suppressor and as an inhibitor of p53 transcriptional activation. The human homologue of this protein is sometimes called Hdm2. The p53 tumor suppressor is the key target of MDM2. It has been identified as a p53 interacting protein that represses p53 transcriptional activity and also acts as an E3 ubiquitin ligase, targeting both itself and p53 for degradation by the proteasome. MDM2 is capable of auto-polyubiquitination, and in complex with p300, a cooperating E3 ubiquitin ligase, is capable of polyubiquitinating p53. Thus a binding ELISA assay was conducted to detect the interaction of recombinant mouse MDM2 and recombinant human P53. Briefly, biotin-linked MDM2 were diluted serially in PBS, with 0.01% BSA (pH 7.4). Duplicate samples of 100μl were then transferred to P53-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST 3 times and incubation with Streptavidin-HRP for 30min, then wells were aspirated and washed 5 times. With the addition of substrate solution, wells were incubated 15-25 minutes at 37℃. Finally, add 50µl stop solution to the wells and read at 450nm immediately. The binding activity of MDM2 and P53 was shown in Figure 1, the EC50 for this effect is 0.064ug/mL.