Analysis of protein ubiquitination.
Attachment of ubiquitin (Ub) to a protein requires a complex of enzymes that recognize the substrate and promote Ub transfer. Sequence motifs present in these enzymes may indicate that other uncharacterized proteins containing these motifs have a biochemical function of Ub-protein ligation, and several in vitro methods are described in this unit for determining if a protein has Ub-transferring activity. They include psmunoblotting of psmunoprecipitated proteins, affinity purification using His-tagged ubiquitin, assaying for auto-ubiquitination of E3, and assaying ubiquitination of a model substrate protein. These methods are suitable for a variety of eukaryotic cells, but techniques are specifically described for use with yeast and mammalian cells.
Laney
Jeffrey D
JD
Yale University, New Haven, Connecticut.
Hochstrasser
Mark
M
Yale University, New Haven, Connecticut.
D000818:Animals; D002846:Chromatography, Affinity; D007150:Immunohistochemistry; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism; D025801:Ubiquitin / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism; D015003:Yeasts / Q000166:cytology / Q000378:metabolism
D011506:Proteins; D025801:Ubiquitin; D044767:Ubiquitin-Protein Ligases