Ubiquitination is a central posttranslational modification that impinges on the fate of proteins. While attachment of K48-linked chains onto soluble proteins marks them for proteolysis via the 26S proteasome, mono-ubiquitination or K63-linked chains result in the endocytosis and sorting through the endomembrane system of integral membrane proteins, such as pattern recognition receptors. In vitro ubiquitination assays allow the biochemical analysis of all individual components of the ubiquitination machinery and its potential substrates. Here, we describe how to reconstitute the ubiquitination cascade in vitro and detail different variations of the assay, the required controls and how to interpret the obtained results.
Keywords: Degradation; Posttranslational modification; Ubiquitination.