Abstract
Ni-NTA affinity chromatography under denaturing conditions has proven to be a powerful method for the isolation of SUMO conjugates from total cell extracts, as it minimizes deconjugation and excludes noncovalent interactions. This chapter describes the use of both His-tagged SUMO and a His-tagged target protein for the characterization of the sumoylation process in the budding yeast Saccharomyces cerevisiae. Two well-studied model substrates, the septin Cdc3 and the replication clamp protein PCNA, are used as examples, but the protocol can easily be adapted to other targets and organisms.
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Acknowledgments
The authors would like to thank E. Johnson for providing the strain CDC3 HA, P. Burgers for plasmid pBL243, and M. Knop for plasmid pYM3. Work in this lab is supported by Cancer Research UK.
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Ulrich, H.D., Davies, A.A. (2009). In Vivo Detection and Characterization of Sumoylation Targets in Saccharomyces cerevisiae . In: Ulrich, H.D. (eds) SUMO Protocols. METHODS IN MOLECULAR BIOLOGY™, vol 497. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-59745-566-4_6
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DOI: https://doi.org/10.1007/978-1-59745-566-4_6
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