Analysis of protein ubiquitination.

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.