GST-N-Myc Ubiquitination Assay with p300

Recombinant GST-N-Myc protein was incubated with or without recombinant p300 protein in a ubiquitination reaction containing 20 mM Hepes (pH 7.5, catalog no.: H3375; Sigma–Aldrich), 5 mM MgCl₂ (catalog no.: AM9530G; Thermo Fisher Scientific), 2 mM dithiothreitol (catalog no.: R0861; Thermo Fisher Scientific), 2 mM ATP (catalog no.: A6559; Sigma–Aldrich), 5 mg of ubiquitin (catalog no.: U5507; Sigma–Aldrich), 20 mM MG132, and 5 μl/reaction crude rabbit reticulocyte lysate (catalog no.: L4151; Promega) for 1 h at 30 °C (24 (link)). Products were then subjected to IP using GST antibody (catalog no.: 2624; Cell Signaling Technology) and protein A/G magnetic beads before Western blot.