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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
An in vitro method for preparing functional glucocorticoid receptor (GR)•hsp90 protein complexes from purified proteins and cellular lysates is described. The method utilizes immunoadsorption of recombinant GR followed by salt-stripping and protein complex reconstitution. The importance of cofactors and buffer conditions are discussed, as are potential method applications.
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
