JoVE Journal

Biochemistry

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

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.

Chapters in this video

0:04

Title

1:04

Site-directed Mutagenesis

2:29

Recombinant Protein Purification and In Vitro Ubiquitination Assay

3:40

Agrobacterium-mediated Transient Protein Expression in Nicotiana benthamiana Leaves and In Planta Ubiquitination Assay

6:36

Establishing the Link Between Enzymatic Activity and Function In Planta

7:27

Results: In Vitro and In Planta Ubiquitination Assay

9:20

Conclusion

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