Analysis of protein ubiquitination

Jeffrey D. Laney, Mark Hochstrasser

Research output: Contribution to journalArticle

16 Scopus citations

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.

Original languageEnglish (US)
Article number14.5
JournalCurrent Protocols in Protein Science
Volume1
Issue numberSUPPL.66
DOIs
StatePublished - Nov 1 2011

Keywords

  • Immunoprecipitation
  • Ubiquitin
  • Ubiquitin-protein ligase

ASJC Scopus subject areas

  • Structural Biology
  • Biochemistry

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