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E3泛素连接酶在哺乳动物细胞裂解物中底物泛素化的评价

JoVE Journal
Bioquímica

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JoVE Journal Bioquímica

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

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E3泛素连接酶在哺乳动物细胞裂解物中底物泛素化的评价

DOI:

10.3791/63561-v

•

09:47 min

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May 10, 2022

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Patrícia M. S. dos Passos*¹, Valentine Spagnol*², Camila R.S.T.B de Correia, Felipe R. Teixeira²

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

Capítulos

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

Tadução automática

我们为哺乳动物细胞中特定底物和E3泛素连接酶的泛素化测定提供了详细的方案。HEK293T细胞系用于蛋白质过表达，通过免疫沉淀从细胞裂解物中纯化多泛型底物，并在SDS-PAGE中分离。免疫印迹用于可视化这种翻译后修饰。

Tags

Keyword Extraction: Substrate Ubiquitylation E3 Ligase Mammalian Cell Lysates Immunoprecipitation Assay Western Blot Human Diseases Cancer Neurological Disorders E3 Ligase Dysregulation HEK293T Cell Line Dulbecco’s Modified Eagles Medium Fetal Bovine Serum Penicillin Streptomycin L-glutamine Trypsin EDTA Polyethyleneimine Opti-MEM 1 Reduced Serum Medium Transient Transfections

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