UNIT 14.5 Analysis of Protein Ubiquitination

  1. Jeffrey D. Laney,
  2. Mark Hochstrasser

Published Online: 1 NOV 2002

DOI: 10.1002/0471140864.ps1405s29

Current Protocols in Protein Science

Current Protocols in Protein Science

How to Cite

Laney, J. D. and Hochstrasser, M. 2002. Analysis of Protein Ubiquitination. Current Protocols in Protein Science. 29:14.5:14.5.1–14.5.11.

Author Information

  1. Yale University, New Haven, Connecticut

Publication History

  1. Published Online: 1 NOV 2002
  2. Published Print: SEP 2002

This is not the most recent version of the article. View current version (1 NOV 2011)


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.