Journal
/
Biochemistry
/
In Vitro Ubiquitination and Deubiquitination Assays of Nucleosomal Histones
JoVE Journal
Biochemistry
A subscription to JoVE is required to view this content.  Sign in or start your free trial.
JoVE Journal Biochemistry
In Vitro Ubiquitination and Deubiquitination Assays of Nucleosomal Histones

In Vitro Ubiquitination and Deubiquitination Assays of Nucleosomal Histones

DOI:
10.3791/59385-v

•

11:36 min

•

July 25, 2019

•

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

Chapters

  • 00:04Title
  • 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

Automatic Translation

Automatic Translation

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

Article

Embed

ADD TO PLAYLIST

Usage Statistics

Related Videos

Automated Acoustic Dispensing for the Serial Dilution of Peptide Agonists in Potency Determination Assays

Reconstitution of Nucleosomes with Differentially Isotope-labeled Sister Histones

Analyzing Supercomplexes of the Mitochondrial Electron Transport Chain with Native Electrophoresis, In-gel Assays, and Electroelution

Small-Scale Colorimetric Assays of Intracellular Lactate and Pyruvate in the Nematode Caenorhabditis elegans

Quantification of Hypopigmentation Activity In Vitro

Isolation of Physiologically Active Thylakoids and Their Use in Energy-Dependent Protein Transport Assays

Detection of Protein Ubiquitination Sites by Peptide Enrichment and Mass Spectrometry

Expression, Purification, Crystallization, and Enzyme Assays of Fumarylacetoacetate Hydrolase Domain-Containing Proteins

Functional Characterization of RING-Type E3 Ubiquitin Ligases In Vitro and In Planta

Stepwise Dosing Protocol for Increased Throughput in Label-Free Impedance-Based GPCR Assays

Read Article