Bu içeriği görüntülemek için JoVE aboneliği gereklidir. Oturum açın veya ücretsiz deneme sürümünü başlatın.
In Vitro Ubiquitination and Deubiquitination Assays of Nucleosomal Histones In Vitro Ubiquitination and Deubiquitination Assays of Nucleosomal Histones In Vitro Ubiquitination and Deubiquitination Assays of Nucleosomal Histones In Vitro
Bölümler
- 00:04Başlık
- 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
L'ubiquitination est une modification post-traductionnelle qui joue un rôle important dans les processus cellulaires et est étroitement coordonnée par la désubiquitination. Les défauts dans les deux réactions sous-tendent les pathologies humaines. Nous fournissons des protocoles pour mener la réaction d'ubiquitination et de deubiquitination in vitro utilisant des composants purifiés.
Etiketler
In Vitro UbiquitinationDeubiquitinationNucleosomal HistonesPurified ComponentsUbiquitination MechanismsDeubiquitination MechanismsEnzyme MutationsHuman DiseaseHigh-throughput ScreeningSmall Molecule InhibitorsTherapeuticsBacteria LysisPMSFAntiprotease CocktailGSH BeadsChromatography ColumnGlutathioneHis-BAP1MBP-DEUBADNickel NTA BeadsImidazole
