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Biochemical Reconstitution of Steroid Receptor•Hsp90 Protein Complexes and Reactivation of Ligand Binding
JoVE Journal
Biology
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JoVE Journal Biology
Biochemical Reconstitution of Steroid Receptor•Hsp90 Protein Complexes and Reactivation of Ligand Binding
DOI:

11:07 min

September 21, 2011

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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

Summary

Automatic Translation

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.

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