Analysis of protein ubiquitination

Jeffrey D. Laney, Mark Hochstrasser

Research output: Contribution to journalArticlepeer-review

21 Scopus citations


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
Issue numberSUPPL.66
StatePublished - Nov 2011
Externally publishedYes


  • Immunoprecipitation
  • Ubiquitin
  • Ubiquitin-protein ligase

ASJC Scopus subject areas

  • Structural Biology
  • Biochemistry


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