A subscription to JoVE is required to view this content. Sign in or start your free trial.
Chapters
- 00:05Title
- 01:37Preparation of Cell Cytosol Containing Functional Glucocorticoid Receptor
- 03:04Immunoabsorption of GR from the Cytosol and Dissociation of hsp90
- 05:16Reconstitution of GR-hsp90 Heterocomplex using Molecular Chaperones, Cofactors, and ATP
- 06:17Analysis of Reconstituted Protein Complexes and Ligand Binding Activity
- 08:26Representative GR-hsp90 Heterocomplex Formation and Steroid Binding Activity
- 10:04Conclusion
En<em> In vitro</em> Metod för att bereda funktionella glukokortikoid-receptorn (GR) • hsp90 proteinkomplex från renade proteiner och cellulära lysates beskrivs. Metoden utnyttjar immunoadsorption av rekombinanta GR följt av salt-strippa och proteinkomplex beredning. Betydelsen av co-faktorerna och villkor buffert diskuteras, liksom möjliga metod applikationer.
Tags
Biochemical ReconstitutionSteroid ReceptorHsp90 Protein ComplexesLigand BindingMolecular ChaperoneEukaryotic Signaling ProteinsTranscription FactorsProtein KinasesCell CyclingTumorigenesisApoptosisSignaling PathwaysGlucocorticoid Receptor (GR)Immunoprecipitation AssayIn Vitro Reconstitution MethodHsp90 Functional ActivitySteroid Receptor Ligand BindingMolecular Chaperone Cofactor Requirements
