Claims
- 1. A method for measuring the presence of a phosphorylated amino acid of a compound by competition, comprising:
a) measuring the fluorescence polarization of a reporter complex comprising a first phosphorylated amino acid bound to a binding molecule; b) adding a substance containing a second phosphorylated amino acid to compete for the binding molecule; c) incubating the solution; d) measuring the fluorescence polarization of the solution during step c); and, e) comparing the fluorescence polarization measurements of step a) with step d).
- 2. The method of claim 1 wherein the compound is a peptide.
- 3. The method of claim 1 wherein the compound is a protein.
- 4. The method of claim 1 wherein the binding molecule comprises a binding protein specific for a phosphorylated amino acid.
- 5. The method of claim 4 wherein the binding protein comprises an antibody specific for a phosphorylated amino acid.
- 6. A method for measuring enzyme activity for attaching and cleaving a phosphate with a compound by competition, comprising:
a) measuring the fluorescence polarization of a reporter molecule comprising a phosphorylated amino acid; b) adding an enzyme and a substrate and, c) incubating the solution; d) measuring the fluorescence polarization of the solution during step c); and, e) comparing the fluorescence polarization measurements of step a) with step d).
- 7. The method of claim 6 wherein the enzyme comprises a kinase for measuring phosphorylation.
- 8. The method of claim 7 wherein a binding protein is added during step c).
- 9. The method of claim 6 wherein the enzyme comprises a phosphatase for measuring phosphate cleavage.
- 10. The method of claim 9 wherein the binding protein is added during step a).
- 11. The method of claim 6 wherein the reporter molecule further comprises a fluorescence-emitting molecule.
- 12. A method for measuring enzyme activity of attaching or cleaving a phosphate and a peptide, comprising:
a) incubating a phosphorylated peptide and an enzyme in solution; b) adding a reporter molecule to the solution; c) measuring the fluorescence polarization of the reporter molecule in step b); d) incubating the solution; e) measuring the fluorescence polarization of the solution during step d); and, f) comparing the fluorescence polarization measurements of step c) with step.
- 13. The method of claim 12 wherein the enzyme comprises a kinase for measuring phosphorylation such that the reporter molecule competes with the peptide when phosphorylated.
- 14. The method of claim 12 wherein the enzyme comprises a phosphatase for measuring phosphate cleavage such that the reporter molecule complex does not compete with the peptide if the phosphate is cleaved.
- 15. A kit utilizing the method of claim 1 for measuring the amount of phosphorylated molecules in a mixture, comprising:
a) instructions for utilizing fluorescence polarization to identify the amount of phosphorylated amino acids in a mixture, b) a receptacle containing a reporter molecule; and, c) a receptacle containing a binding protein.
- 16. The kit of claim 15 wherein the reporter molecular complex comprises a fluorescence-emitting peptide.
- 17. The kit of claim 16 wherein the binding protein comprises an antibody specific for phosphorylated amino acids.
- 18. The kit of claim 17 wherein the enzyme comprises kinase.
- 19. The kit of claim 18 wherein the enzyme comprises phosphatase.
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This is a Provisional Application Ser. No. 60/029,831, filing date Oct. 28, 1996.