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In Vitro Ubiquitination and Deubiquitination Assays of Nucleosomal Histones
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JoVE Journal Biochimica
In Vitro Ubiquitination and Deubiquitination Assays of Nucleosomal Histones

In Vitro Ubiquitination and Deubiquitination Assays of Nucleosomal Histones

DOI:
10.3791/59385-v

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11:36 min

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July 25, 2019

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, , , , , , , ,
1Maisonneuve-Rosemont Hospital Research Center and Department of Medicine,University of Montréal, 2Lunenfeld-Tanenbaum Research Institute,Sinai Health System

Capitoli

  • 00:04Titolo
  • 01:01GSH-agarose Affinity Purification of the GST-RING1B(1-159)-BMI1 (1-109) E3 Ubiquitin Ligase Complex
  • 02:16Purification of the BAP1/DEUBAD (ASXL2) Deubiquitinase Complex
  • 04:58Purification of the Nucleosomes from HEK293T Cells
  • 07:38Nucleosome Ubiquitination Assay Using BMI1/RING1B E3 Ubiquitin Ligase Complex and In Vitro Nucleosomal H2A DUB Assay Using BAP1/DEUBAD
  • 09:05Results: Purification, Ubiquitination, and Deubiquitination
  • 10:40Conclusion

Summary

Traduzione automatica

Traduzione automatica

Ubiquitination is a post-translational modification that plays important roles in cellular processes and is tightly coordinated by deubiquitination. Defects in both reactions underlie human pathologies. We provide protocols for conducting ubiquitination and deubiquitination reaction in vitro using purified components.

Tags

In Vitro UbiquitinationDeubiquitinationNucleosomal HistonesPurified ComponentsUbiquitination MechanismsDeubiquitination MechanismsEnzyme MutationsHuman DiseaseHigh-throughput ScreeningSmall Molecule InhibitorsTherapeuticsBacteria LysisPMSFAntiprotease CocktailGSH BeadsChromatography ColumnGlutathioneHis-BAP1MBP-DEUBADNickel NTA BeadsImidazole

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