Mdm2 p53 ubiquitination kit

We performed In vitro ubiquitination assay utilizing MDM2/p53 Ubiquitination Kit (Boston Biochem). The purified His-tagged HBP1 and E1 enzyme, E2 enzyme, GST-MDM2, ubiquitin, Mg2+-ATP were incubated with Reaction Buffer 1 h at 30 °C water bath, and the mixture were subsequently separated by SDS-PAGE and analyzed by western blotting or Coomassie blue staining for mass spectrometry.

Reactions were prepared on ice in 0.5 ml polypropylene tubes using the MDM2/p53 Ubiquitination Kit (Boston Biochem) according to the manufacturer’s protocol. The reaction included 9 µl H₂O and 3 µl each of the following (10× solutions): reaction buffer, p53 substrate protein, E1 enzyme, E2 enzyme, MDM2 enzyme, Mg²⁺-ATP. Plates were gently mixed and the reaction was started by adding 3 µl 10× Ubiquitin solution. After incubation for 1 h at 37 °C, SDS-PAGE sample buffer containing 1.0 M DTT was added and samples were heat denatured at 98 °C for 5 min. Two control reactions (one with ATP omitted and another with 80 nM Nutlin 3) were run simultaneously. To determine the effect of PC₁₆ on the p53 ubiquitination, reactions were performed in the presence of increasing concentrations of ceramide (60, 80, and 100 nM). After denaturing, samples were analyzed by SDS-PAGE followed by WB using the manufacturer supplied p53 antibody.