Analysis of Protein Ubiquitination

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