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. © 2011 by John Wiley & Sons, Inc.
CITATION STYLE
Laney, J. D., & Hochstrasser, M. (2011). Analysis of protein ubiquitination. Current Protocols in Protein Science, 1(SUPPL.66). https://doi.org/10.1002/0471140864.ps1405s66
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