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.
Ulrich
Helle D
HD
Cancer Research UK London Research Institute, Clare Hall Laboratories, South Mimms, UK.
Davies
Adelina A
AA
D019411:Clinical Laboratory Techniques; D003001:Cloning, Molecular / Q000379:methods; D008954:Models, Biological; D030781:Organisms, Genetically Modified; D011499:Protein Processing, Post-Translational; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D025841:Small Ubiquitin-Related Modifier Proteins / Q000302:isolation & purification / Q000378:metabolism*
D029701:Saccharomyces cerevisiae Proteins; D025841:Small Ubiquitin-Related Modifier Proteins