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Functional Characterization of RING-Type E3 Ubiquitin Ligases In Vitro and In Planta
JoVE Journal
Biochemistry
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JoVE Journal Biochemistry
Functional Characterization of RING-Type E3 Ubiquitin Ligases In Vitro and In Planta

Functional Characterization of RING-Type E3 Ubiquitin Ligases In Vitro and In Planta

DOI:
10.3791/60533-v

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10:27 min

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December 05, 2019

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, , , , , ,
1Department of Entomology, Plant Pathology and Nematology,University of Idaho, 2Department of Plant Sciences,University of Idaho

Chapters

  • 00:04Title
  • 01:04Site-directed Mutagenesis
  • 02:29Recombinant Protein Purification and In Vitro Ubiquitination Assay
  • 03:40Agrobacterium-mediated Transient Protein Expression in Nicotiana benthamiana Leaves and In Planta Ubiquitination Assay
  • 06:36Establishing the Link Between Enzymatic Activity and Function In Planta
  • 07:27Results: In Vitro and In Planta Ubiquitination Assay
  • 09:20Conclusion

Summary

Automatic Translation

Automatic Translation

The goal of this manuscript is to present an outline for the comprehensive biochemical and functional studies of the RING-type E3 ubiquitin ligases. This multistep pipeline, with detailed protocols, validates an enzymatic activity of the tested protein and demonstrates how to link the activity to function.

Tags

Keywords: RING-type E3 Ubiquitin LigasesSite-directed MutagenesisIn Vitro Enzymatic ActivityIn Planta FunctionalityUbiquitination AssayRING DomainCys And His ResiduesPCRDpn1 DigestionPMAL-c2 VectorMBP TagSDS-PAGEPVDF MembraneWestern Blotting

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