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Detection of protein SUMOylation in vivo

Abstract

The small ubiquitin-like modifiers (SUMOs) are posttranslationally conjugated to eukaryotic cellular proteins with generally unpredictable consequences. SUMO substrates are found in many cellular systems, and functional analysis has revealed that substrate SUMOylation often has an important role in their regulation. Here we describe a cell-based protocol which can be used to detect the SUMOylation of a protein that relies on the enrichment of SUMO conjugates by purification of 6His-SUMO under denaturing conditions, followed by western blotting for the protein of interest. By purifying under denaturing conditions this method not only reduces the risk of false-positive identifications by non-covalent interactions, but also preserves SUMO-substrate conjugates by inhibiting SUMO-specific proteases—two caveats that may complicate other less stringent purification methods. In preliminary form, this protocol takes 4–5 d to perform, and it can be further elaborated to provide a multi-angled approach to investigate protein conjugation by SUMO.

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Figure 1: Stable expression of the three 6His-small ubiquitin-like modifier (SUMO) paralogues in HeLa cells.
Figure 2: Workflow of a typical in vivo analysis for protein SUMOylation for one test protein.
Figure 3: Results of an experiment to determine if a test protein is modified by small ubiquitin-like modifier-1 (SUMO-1) or SUMO-2 in vivo.

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Acknowledgements

M.H.T. is supported by a Cancer research UK program grant, and M.S.R. is funded by the Ramón y Cajal Program, Ministerio de Educación y Ciencia grant BFU 2005-04091, Fondo de Investigaciones Sanitarias, CIBERhed. D.P.X. is a fellow of the Association for International Cancer Research.

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Contributions

M.H.T. was involved in the development of the protocol and prepared the manuscript. M.S.R. created the 6His-SUMO cell lines, devised and refined the purification technique and edited the manuscript. D.P.X. was involved in developing and refining the technique and edited the manuscript. R.T.H. is the principal investigator that supervised the work and edited the manuscript.

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Correspondence to Michael H Tatham.

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Tatham, M., Rodriguez, M., Xirodimas, D. et al. Detection of protein SUMOylation in vivo. Nat Protoc 4, 1363–1371 (2009). https://doi.org/10.1038/nprot.2009.128

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