title: Analysis of protein ubiquitination.
abstract: Attachment of ubiquitin (Ub) to a protein requires a series of enzymes that recognize the substrate and promote Ub transfer. Several methods are described in this unit for determining if a protein has Ub-transferring activity. They include immunoblotting of immunoprecipitated proteins, affinity purification using His-tagged Ub, assaying for auto-ubiquitination of E3, and assaying ubiquitination of a model substrate protein in vitro and in E. coli cells that express Ub-ligation enzymes. These methods are suitable for a variety of eukaryotic cells, but techniques are specifically described for use with yeast and mammalian cells.
authors: Department of Molecular Biology, Cell Biology, and Biochemistry, Brown University, Providence, Rhode Island.
authors: Jeffrey D
authors: JD
authors: Laney
authors: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut.
authors: Mark
authors: M
authors: Hochstrasser
mesh_terms: D000818:Animals; D002846:Chromatography, Affinity / Q000379:methods; D005656:Fungal Proteins / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting / Q000379:methods*; D047468:Immunoprecipitation / Q000379:methods*; D011506:Proteins / Q000378:metabolism*; D025801:Ubiquitin / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism*; D054875:Ubiquitination; D015003:Yeasts / Q000378:metabolism
chemical_list: D005656:Fungal Proteins; D011506:Proteins; D025801:Ubiquitin; D044767:Ubiquitin-Protein Ligases