Monitoring HECT Ubiquitination Activity In Vitro

In vitro ubiquitination tools have been employed to mechanistically study the ubiquitin enzymatic cascade. Here, we describe an assay capable to monitor ubiquitin conjugation in real time using the Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) system. The assay requires purified E1 and E2 enzymes, the HECT E3 ligase of choice and two fluorophore-labeled ubiquitins. This single step technique represents an excellent tool to study the enzymatic activity during chain elongation, to compare ligase activity in the presence or absence of the substrate, and to set-up high-throughput screenings for enzymatic activity modulators (i.e., activators or inhibitors).