In Vivo Detection and Characterization of Sumoylation Targets in Saccharomyces cerevisiae

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.