Assessing Ubiquitylation of Individual Proteins Using Xenopus Extract Systems

Xenopus extract systems have been used to study ubiquitylation of proteins, and to uncover some of the fundamental processes of the ubiquitylation pathway itself. They provide a simple, quick, and robust method for studying ubiquitylation. In this protocol, methods are provided for studying protein ubiquitylation using Xenopus egg or embryo extracts and in vitro radiolabeled proteins. These methods also enable examination of whether proteins undergo noncanonical ubiquitylation, through modification of the protein by covalent linkage to ubiquitin through residues other than lysine, such as cysteine, serine, and threonine.