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

09:47 min

May 10, 2022

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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

Automatic Translation

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.

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