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Evaluation of Substrate Ubiquitylation by E3 Ubiquitin-ligase in Mammalian Cell Lysates
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Biochemistry
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JoVE Journal Biochemistry
Evaluation of Substrate Ubiquitylation by E3 Ubiquitin-ligase in Mammalian Cell Lysates

Evaluation of Substrate Ubiquitylation by E3 Ubiquitin-ligase in Mammalian Cell Lysates

DOI:
10.3791/63561-v

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09:47 min

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May 10, 2022

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, , ,
1Department of Genetic and Evolution,Federal University of São Carlos, 2Department of Biochemistry and Immunology, Faculty of Medicine of Ribeirão Preto,University of São Paulo

Chapters

  • 00:05Introduction
  • 01:08Cell Culture
  • 02:33Cell Transfection
  • 03:41Cell Lysis and Immunoprecipitation
  • 07:40Results: Specific Polyubiquitylation of UXT-V2-HA Mediated by Fbxo7
  • 09:06Conclusion

Summary

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We provide a detailed protocol for a ubiquitylation assay of a specific substrate and an E3 ubiquitin-ligase in mammalian cells. HEK293T cell lines were used for protein overexpression, the polyubiquitylated substrate was purified from cell lysates by immunoprecipitation, and resolved in SDS-PAGE. Immunoblotting was used to visualize this post-translational modification.

Tags

Keyword Extraction:Substrate UbiquitylationE3 LigaseMammalian Cell LysatesImmunoprecipitation AssayWestern BlotHuman DiseasesCancerNeurological DisordersE3 Ligase DysregulationHEK293T Cell LineDulbecco’s Modified Eagles MediumFetal Bovine SerumPenicillinStreptomycinL-glutamineTrypsin EDTAPolyethyleneimineOpti-MEM 1 Reduced Serum MediumTransient Transfections

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