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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

Article DOI: 10.3791/63561-v • 09:47 min • May 10th, 2022
May 10th, 2022 •
*1, *2, 1, 1,2
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
* These authors contributed equally

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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

Substrate Ubiquitylation E3 Ubiquitin-ligase Mammalian Cell Lysates Ligase-substrate Interaction Immunoprecipitation Assay Western Blot Plasmids Gene Coding Cancer Neurological Disorders Disease Treatment Disease Diagnosis Valentine Spagnol HEK293T Cell Line Dulbecco's Modified Eagles Medium Fetal Bovine Serum Penicillin Streptomycin L-glutamine Trypsin Ethylenediaminetetraacetic Acid Solution
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