Preparation, characterization, and use of tagged ubiquitins

The low abundance and heterogeneity of ubiquitinated proteins has led to the development and use of tagged forms of ubiquitin. Additional residues present at the ubiquitin amino terminus provide immunological and/or affinity sites to facilitate visualization, identification, and purification of ubiquitinated substrates by virtue of their covalent attachment to the tagged ubiquitin. The use of tagged ubiquitin to understand the scope, nature, and biological relevance of this conserved modification system has been demonstrated in multiple ways. Unknown substrates can be identified, or a previously identified substrate can be analyzed with tagged ubiquitin in vitro or in vivo to determine the specificity, regulation, and type of ubiquitin linkages formed. This contribution describes the generation and use of multiple types of modified ubiquitins: biotinylated ubiquitin produced in vitro, or GST-, myc-, HA-, and hexahistidine-tagged ubiquitins produced in vivo.