Experimental tests to definitively determine ubiquitylation of a substrate.

Ubiquitin-mediated proteolysis is a major pathway of protein degradation that regulates numerous cellular processes. An understanding of the circumstances that contribute to the ubiquitylation of a specific protein can yield vast insight into its regulation. This article examines multiple procedures that explain whether a protein is ubiquitylated and suggests methods to investigate the factors that specifically target the substrate for ubiquitylation, as well as the site of ubiquitin conjugation.