title: In vitro protein ubiquitination assay.
abstract: Ubiquitination is one of the most important posttranslational modifications in all eukaryote organisms. Ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), and ubiquitin ligase (E3) are the three key enzymes in this process. To detect the specificity between E2 and E3 or enzyme-substrate relationship between E3 and a substrate protein, ubiquitination activity needs to be determined. This protocol provides a convenient and efficient in vitro assay for DTT-sensitive thioester formation of E2s and Ring/U-box-type E3s, and E3-mediated substrate ubiquitination. E2/E3 specificities can also be investigated quickly by using this system. This method can be applied to ubiquitination assays of proteins from any eukaryotic organisms.
authors.affiliation: State Key Laboratory of Plant Genomics, National Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China.
authors.forename: Qingzhen
authors.initials: Q
authors.lastname: Zhao
authors.forename: Lijing
authors.initials: L
authors.lastname: Liu
authors.forename: Qi
authors.initials: Q
authors.lastname: Xie
mesh_terms: D004926:Escherichia coli / Q000378:metabolism; D008025:Ligases / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D044763:Ubiquitin-Conjugating Enzymes / Q000235:genetics / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism; D054875:Ubiquitination
chemical_list: D044763:Ubiquitin-Conjugating Enzymes; D044767:Ubiquitin-Protein Ligases; D008025:Ligases