Fluorescent ubiquitin

Different prepared derivatives were evaluated for their inhibitory potential against p53 ubiquitination. The method is based on incubating different compounds with GST-tagged HDM2, immobilized on glutathione-Sepharose, p53, ubiquitin, E1 and E2 (UbcH5B) ligases. The buffer contained ATP as detailed by Gomha and Abdelaziz [44 (link)]. The reaction products were then resolved by SDSPAGE and p53 ubiquitination and were quantified by Western blotting using an anti-p53 antibody [45 ].
Briefly, rGSH-S-transferase-tagged MDM2 (GST-MDM2) was expressed in Escherichia coli BL21 cells using 1 mM isopropyl-thio-b-d-galactoside for 3 h. The enzyme was then purified on glutathione-sepharose beads (Amersham). Beads were washed with Tris (pH 7.5). The reaction contained fluorescent ubiquitin (5 µg; Invitrogen), 50 ng mammalian E1 (Enzo), 200 ng human rUbcH5B E2 (Enzo), 200 ng His-p53 (Enzo) and buffer [50 mM Tris pH 8,2 mM dithiothreitol, 5 mM MgCl₂, 2 mM adenosine triphosphate (ATP)]. The MPD-compound or control mixture was dose-titrated onto GS4b-MDM2 beads. Incubation at 37 °C was followed by shaking (1200 rpm, 1 h). The reaction was terminated with 3× sodium dodecyl sulfate sample buffer. Finally, unbound fluorescent ubiquitin was drained and the total fluorescent ubiquitin signal was measured using a plate reader (Safire).