title: Analysis of cellular SUMO and SUMO-ubiquitin hybrid conjugates.
abstract: Posttranslational modification of proteins with the small ubiquitin-related modifier (SUMO) has been implicated in many important physiological functions, including the regulation of transcription and DNA repair. In most cases, only a small fraction of the total cellular amounts of a given protein is sumoylated at a certain point in time. Sensitive detection of sumoylated forms of proteins by western blotting is, therefore, an important step in the identification and/or characterization of a protein control by sumoylation. Polysumoylated proteins are recognized and targeted to the proteasome by specific ubiquitin ligases bearing SUMO interaction motifs. Sumoylation itself is reversible by the action of desumoylating enzymes. Their activities cause a rapid loss of SUMO conjugates in most standard cell extracts. To preserve SUMO-protein conjugates, therefore, a preparation of extracts under denaturing conditions is recommended. Here, we describe the application of an alkaline lysis procedure and a western blot protocol for the analysis of SUMO conjugates in yeast and human cells. In addition, we describe the application of another extraction procedure combined with immobilized metal affinity chromatography for the analysis of ubiquitin-SUMO hybrid conjugates from yeast and human cells.
authors.affiliation: Institute for Genetics, University of Cologne, Zülpicher Str. 47a, 50674 Cologne, Germany.
authors.forename: Marion
authors.initials: M
authors.lastname: Schnellhardt
authors.forename: Kristina
authors.initials: K
authors.lastname: Uzunova
authors.forename: Veronika N
authors.initials: VN
authors.lastname: Bade
authors.forename: Anke
authors.initials: A
authors.lastname: Krause
authors.forename: Stefan R
authors.initials: SR
authors.lastname: Weisshaar
authors.forename: Gerrit J K
authors.initials: GJ
authors.lastname: Praefcke
authors.forename: R Jürgen
authors.initials: RJ
authors.lastname: Dohmen
mesh_terms: D015153:Blotting, Western / Q000379:methods; D002846:Chromatography, Affinity / Q000379:methods; D006801:Humans; D012441:Saccharomyces cerevisiae; D025841:Small Ubiquitin-Related Modifier Proteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism*; D058207:Sumoylation; D025801:Ubiquitin / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism*; D044763:Ubiquitin-Conjugating Enzymes / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism
chemical_list: D025841:Small Ubiquitin-Related Modifier Proteins; D025801:Ubiquitin; D044763:Ubiquitin-Conjugating Enzymes; D044767:Ubiquitin-Protein Ligases