Claims
- 1. A method of determining whether a fluorescently labeled compound is phosphorylated, the method comprising:
providing the compound in a mixture with a polymer having multivalent metal cations associated therewith which polymer is sufficiently large to induce a shift in an amount of polarized fluorescence emitted from the mixture when the multivalent metal cations bind the polymer to the compound; and monitoring the amount of polarized fluorescence emitted from the mixture to determine whether the multivalent metal cations bind the polymer to the compound, such binding being indicative that the compound is phosphorylated.
- 2. The method of claim 1, wherein the multivalent metal cations are selected from the group consisting of Fe3+, Ca2+, Ni2+ and Zn2+.
- 3. The method of claim 1, wherein the multivalent metal cations are chelated to the polymer.
- 4. The method of claim 1, wherein the polymer is between 5 kD and 1000 kD.
- 5. The method of claim 1, wherein prior to the providing step, the compound is contacted with an enzyme that either adds or removes a phosphate group to or from the compound under conditions suitable for addition or removal of a phosphate group, respectively.
- 6. The method of claim 5, wherein prior to the providing step, the compound is contacted with a kinase enzyme in the presence of a phosphate donor group.
- 7. The method of claim 5, wherein prior to the providing step, the compound is contacted with a phosphatase enzyme that removes a phosphate group from the compound.
- 8. The method of claim 5, wherein prior to the providing step, the compound is contacted with an enzyme that either adds or removes a phosphate group to or from the compound, in the presence of at least a first test compound.
- 9. The method of claim 1, wherein the compound comprises a polypeptide.
- 10. The method of claim 1, wherein the compound comprises a serine, tyrosine, or threonine substrate.
- 11. The method of claim 1, wherein the multivalent metal cations bind to the phosphorylated compound based at least partially on a difference in charge between the phosphorylated compound and the multivalent metal cations.
- 12. The method of claim 11, wherein the multivalent metal cations bind to the phosphorylated compound based at least partially on an affinity between the metal cations and a phosphate group associated with the phosphorylated compound.
- 13. The method of claim 1, wherein the multivalent metal cations bind to the phosphorylated compound based at least partially on an affinity between the metal cations and a phosphate group associated with the phosphorylated compound.
- 14. The method of claim 1, wherein the multivalent metal cations comprise trivalent metal cations.
- 15. The method of claim 14, wherein the trivalent metal cations comprise Fe3+.
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application is a continuation of U.S. patent application Ser. No. 09/865,044, filed May 24, 2001, which is a continuation of U.S. patent application Ser. No. 09/316,447, filed May 21, 1999, now U.S. Pat. No. 6,287,774, each of which are incorporated herein in its entirety for all purposes.
Continuations (2)
|
Number |
Date |
Country |
Parent |
09865044 |
May 2001 |
US |
Child |
10397887 |
Mar 2003 |
US |
Parent |
09316447 |
May 1999 |
US |
Child |
09865044 |
May 2001 |
US |