UNIT 14.5 Analysis of Protein Ubiquitination

  1. Jeffrey D. Laney1,
  2. Mark Hochstrasser2

Published Online: 1 NOV 2011

DOI: 10.1002/0471140864.ps1405s66

Current Protocols in Protein Science

Current Protocols in Protein Science

How to Cite

Laney, J. D. and Hochstrasser, M. 2011. Analysis of Protein Ubiquitination. Current Protocols in Protein Science. 66:14.5:14.5.1–14.5.13.

Author Information

  1. 1

    Department of Molecular Biology, Cell Biology, and Biochemistry, Brown University, Providence, Rhode Island

  2. 2

    Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut

Publication History

  1. Published Online: 1 NOV 2011
  2. Published Print: NOV 2011


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. Curr. Protoc. Protein Sci. 66:14.5.1-14.5.13. © 2011 by John Wiley & Sons, Inc.


  • ubiquitin;
  • immunoprecipitation;
  • ubiquitin-protein ligase