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In Vitro Analysis of E3 Ubiquitin Ligase Function
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JoVE Journal Biology
In Vitro Analysis of E3 Ubiquitin Ligase Function

In Vitro Analysis of E3 Ubiquitin Ligase Function

DOI:
10.3791/62393-v

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06:06 min

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May 14, 2021

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, , ,
1Institute for Genetics and Cologne Excellence Cluster on Cellular Stress Responses in Aging Associated Diseases (CECAD),University of Cologne, 2Center for Molecular Medicine Cologne,University of Cologne

Chapters

  • 00:05Introduction
  • 00:43In Vitro Auto-Ubiquitylation Assay
  • 01:31In vitro Substrate Ubiquitylation Assay
  • 02:13Lysine Discharge Assay
  • 03:37Results: Analysis of the Ubiquitylation Products
  • 05:28Conclusion

Summary

Automatic Translation

Automatic Translation

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.

Tags

E3 Ubiquitin LigaseIn Vitro AnalysisE2 Enzyme SpecificitySubstrate SelectionUbiquitin TransferAuto-ubiquitylation AssaySubstrate Ubiquitylation AssayLysine Discharge AssayRecombinant ExpressionEukaryotic ProteinsSDS-PAGEThermal CyclerMaster MixEDTAApyrase

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