The overall goal of this protocol is to analyze important key aspects of E3 ligase function, including E2 enzyme specificity, substrate selection and ubiquitin transfer. The main advantage of this technique is its wide applicability, as proteins f
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The present study provides detailed in vitro ubiquitylation assay protocols for the analysis of E3 ubiquitin ligase catalytic activity. Recombinant proteins were expressed using prokaryotic systems such as Escherichia coli culture.