A Fluorescence‐Based Assay to Monitor SUMOylation in Real‐Time

Abstract

The small ubiquitin‐like modifier (SUMO) is an important post‐translational modifier that regulates various cellular processes. Extensive investigations have been made to comprehend the enzymatic process and consequence of SUMOylation. In vitro SUMOylation assays are invaluable for understanding the fundamental mechanisms of SUMOylation. A majority of these assays monitor changes in the size of the substrate upon SUMO conjugation. Current methods typically detect the size difference through SDS‐PAGE and western blots, which makes these methods cumbersome, error‐prone, and time‐consuming. Here, we describe a fluorescence‐based assay for real‐time detection of SUMOylation. In the method, a fluorophore‐tagged substrate is used in the SUMOylation reaction. Upon SUMOylation, the size and correlation time (τc) of the substrate increases, and so does its anisotropy. The rate of change in anisotropy with time reflects the efficiency of the SUMOylation machinery. The real‐time SUMOylation assay protocol is elegant, time‐saving, and less prone to errors. © 2020 Wiley Periodicals LLC. Basic Protocol: Fluorescent anisotropy‐based in vitro SUMOylation assay

The Next Frontier: Translational Development of Ubiquitination, SUMOylation, and NEDDylation in Cancer

“…Alternatively, SUMOylation of a purified recombinant protein of interest or 35 S-labeled translation product, reconstituted in an in vitro SUMOylation enzymatic reaction, can be detected using Western blotting or autoradiography, respectively [ 20 ]. Similar to ubiquitination, SUMOylation can also be monitored in real time using either a fluorophore-tagged substrate or SUMO protein in a SUMOylation reaction [ 178 , 179 ]. A FRET-based assay was developed using yellow fluorescent protein (YFP)-tagged mature SUMO-1 and cyan fluorescent protein (CFP)-tagged Ran GTPase-activating protein 1 (RanGAP1) and its utility has been demonstrated in a high-throughput screening effort.…”