Claims
- 1. A fluorescence polarization assay for determining whether a compound binds steroid receptor comprising:
(a) determining the fluorescence polarization values of a free fluorescently-labeled probe and the fluorescently-labeled probe bound to an expression vector lysate, wherein the lysate comprises a steroid receptor associated with at least three heat shock proteins (hsps) to obtain a range of fluorescence polarization values and selecting a reference fluorescence polarization value falling within that range; (b) mixing the fluorescently-labeled probe with the lysate in step (a) in a buffered aqueous solution; (c) mixing a test compound with the mixture obtained in step (b) and incubating the resulting mixture of fluorescently-labeled probe, lysate, and test compound; (d) measuring the fluorescence polarization value of the incubated mixture obtained in step (c) to obtain a test fluorescence polarization value; and (e) determining the difference between the test fluorescence polarization value and the reference fluorescence polarization value; wherein the difference in fluorescence polarization values obtained in step (e) indicates whether the test compound binds the steroid receptor.
- 2. The method according to claim 1, wherein the probe is dexamethasone or mifepristone.
- 3. The method according to claim 1 wherein the fluorescent label is rhodamine or a rhodamine derivative.
- 4. The method according to claim 1, wherein the fluorescently-labeled probe is tetramethylrhodamine-dexamethasone.
- 5. The method according to claim 1, wherein the fluorescently-labeled probe is tetramethylrhodamine-mifepristone.
- 6. The method according to claim 1, wherein the expression vector is selected from viral, yeast or human cell lines.
- 7. The method according to claim 1, wherein the expression vector is a baculovirus system.
- 8. The method according to claim 1, wherein the steroid receptor is selected from the group consisting of progesterone receptor, glucocorticoid receptor, and mineralocorticoid receptor.
- 9. The method according to claim 1, wherein the steroid receptor is glucocorticoid receptor.
- 10. The method according to claim 1, wherein the heat shock protein is selected from the group consisting of hsp90, hsp70, hsp60 and hsp23.
- 11. The fluorescently-labeled probe according to claim 1, wherein the emission of said fluorescent label is at least about 450 nm to 700 nm.
- 12. The fluorescently-labeled probe according to claim 1, wherein the emission of said fluorescent label is at least about 550 nm to 700 nm.
- 13. The method according to claim 1 wherein the range of fluorescence polarization values in step (a) is obtained by periodically adding increasing amounts of expression vector lysate.
- 14. The method according to claim 13 wherein the increasing amounts of expression vector lysate is added until no further significant change in polarization value is observed.
- 15. A fluorescence polarization assay for determining whether a compound binds steroid receptor comprising a library of test compounds comprises the following steps:
(a) determining the fluorescence polarization values of a free fluorescently-labeled probe and the fluorescently-labeled probe bound to an expression vector lysate wherein the lysate comprises a steroid receptor associated with at least three heat shock proteins (hsps) to obtain a range of fluorescence polarization values and selecting a reference fluorescence polarization value falling within that range; (b) mixing the fluorescently-labeled probe with the lysate in step (a) in a buffered aqueous solution; (c) adding test compounds to a plurality of containers; (d) adding the mixture obtained in step (b) to said plurality of containers, and incubating the resulting mixtures of fluorescently-labeled probe, lysate, and test compounds; (e) measuring the fluorescence polarization values of the incubated mixtures obtained in step (d) to obtain test fluorescence polarization values; and (f) determining the differences between the test fluorescence polarization values and the reference fluorescence polarization value; wherein the differences in fluorescence polarization values obtained in step (f) indicate whether the test compounds bind steroid receptor.
- 16. The method according to claim 15, wherein the probe is dexamethasone or mifepristone.
- 17. The method according to claim 15, wherein the fluorescent label is rhodamine or a rhodamine derivative.
- 18. The method according to claim 15, wherein the fluorescently-labeled probe is tetramethylrhodamine-dexamethasone.
- 19. The method according to claim 15, wherein the fluorescently-labeled probe is tetramethylrhodamine-mifepristone.
- 20. The method according to claim 15, wherein the expression vector is selected from viral, yeast or human cell lines.
- 21. The method according to claim 15, wherein the expression vector is a baculovirus system.
- 22. The method according to claim 15, wherein the steroid receptor is selected from the group consisting of progesterone receptor, glucocorticoid receptor, and mineralocorticoid receptor.
- 23. The method according to claim 15, wherein the steroid receptor is glucocorticoid receptor.
- 24. The method according to claim 15, wherein the heat shock protein is selected from the group consisting of hsp90, hsp70, hsp60 and hsp23.
- 25. The fluorescently-labeled probe according to claim 15, wherein the emission of said fluorescent label is at least about 450 nm to 700 nm.
- 26. The fluorescently-labeled probe according to claim 15, wherein the emission of said fluorescent label is at least about 550 nm to 700 nm.
- 27. The method according to claim 15, wherein the range of fluorescence polarization values in step (a) is obtained by periodically adding increasing amounts of expression vector lysate.
- 28. The method according to claim 27 wherein the increasing amounts of expression vector lysate is added until no further significant change in polarization value is observed.
- 29. A fluorescence polarization assay for determining whether a compound binds steroid receptor comprising:
(a) determining the fluorescence polarization values of a free fluorescently-labeled probe and the fluorescently-labeled probe bound to an expression vector lysate, wherein the lysate comprises a steroid receptor associated with at least three heat shock proteins (hsps) to obtain a range of fluorescence polarization values and selecting a reference fluorescence polarization value falling within that range; (b) mixing the fluorescently-labeled probe with the lysate in step (a) in a buffered aqueous solution; (c) mixing a test compound dissolved in a buffered aqueous solution with the mixture obtained in step (b) and incubating the resulting mixture of fluorescently-labeled probe, lysate, and test compound; (d) measuring the fluorescence polarization value of the incubated mixture obtained in step (c) to obtain a test fluorescence polarization value; and (e) determining the difference between the test fluorescence polarization value and the reference fluorescence polarization value; wherein the difference in fluorescence polarization values obtained in step (e) indicates whether the test compound binds the steroid receptor.
- 30. The method according to claim 29, wherein the probe is dexamethasone or mifepristone.
- 31. The method according to claim 29, wherein the fluorescent label is rhodamine or a rhodamine derivative.
- 32. The method according to claim 29, wherein the fluorescently-labeled probe is tetramethylrhodamine-dexamethasone.
- 33. The method according to claim 29, wherein the fluorescently-labeled probe is tetramethylrhodamine-mifepristone.
- 34. The method according to claim 29, wherein the expression vector is selected from viral, yeast or human cell lines.
- 35. The method according to claim 29, wherein the expression vector is a baculovirus system.
- 36. The method according to claim 29, wherein the steroid receptor is selected from the group consisting of progesterone receptor, glucocorticoid receptor, and mineralocorticoid receptor.
- 37. The method according to claim 29, wherein the steroid receptor is glucocorticoid receptor.
- 38. The method according to claim 29, wherein the heat shock protein is selected from the group consisting of hsp90, hsp70, hsp60 and hsp23.
- 39. The fluorescently-labeled probe according to claim 29, wherein the emission of said fluorescent label is at least about 450 nm to 700 nm.
- 40. The fluorescently-labeled probe according to claim 29, wherein the emission of said fluorescent label is at least about 550 nm to 700 nm.
- 41. The method according to claim 29, wherein the range of fluorescence polarization values in step (a) is obtained by periodically adding increasing amounts of expression vector lysate.
- 42. The method according to claim 41, wherein the increasing amounts of expression vector lysate is added until no further significant change in polarization value is observed.
RELATED APPLICATIONS
[0001] This application claims priority benefit to U.S. Provisional Application serial No. 60/291,877 filed May 18, 2001.
Provisional Applications (1)
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Number |
Date |
Country |
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60291877 |
May 2001 |
US |