Functional analysis of protein ubiquitination

Alterations in the protein ubiquitination can lead to the development of serious pathological conditions and diseases and, therefore, are under extensive investigation. Here we detail the revised/updated version of two approaches for analyzing the functional activities of the ubiquitin transferring system and target protein ubiquitination. These approaches permit the analysis of protein ubiquitination within the cellular environment as well as in a tube when the purified components are used. The updates introduced in the protocols allow both to increase the sensitivity of the assays and to reduce the false positives often experienced in the analyses.