Claims
- 1. A method of assaying an enzyme-mediated coupling reaction between a first and a second reactant, comprising:
contacting the first reactant with the second reactant in the presence of the enzyme wherein the second reactant comprises a thiol derivative, to yield a first product comprising the thiol derivative, and further wherein the first reactant comprises a fluorescent label, the fluorescent label being incorporated into the first product; contacting the first product with a third reactant, the third reactant comprising a thiol reactive derivative; the thiol reactive derivative being incorporated into a second product; and adding a fourth reactant to the second product, the fourth reactant comprising a large molecular weight moiety, and measuring a difference in a fluorescence polarization level from the second product as compared to the fluorescence polarization of the first reactant.
- 2. The method of claim 1, wherein the thiol reactive derivative comprises a biotin derivative.
- 3. The method of claim 2, wherein the biotin derivative comprises biotin-HPDP.
- 4. The method of claim 3, wherein the biotin derivative comprises a biotin haloacetate.
- 5. The method of claim 3, wherein the biotin derivative comprises biotin maleamide group.
- 6. The method of claim 1, wherein the contacting the first product with the third reactant step further comprises adjusting pH to within about 2 to about 8.
- 7. The method of claim 6, wherein the pH is adjusted to within about 3.5 to about 7.5.
- 8. The method of claim 6, wherein the pH is adjusted to within about 4.0 to about 5.0.
- 9. The method of claim 6, wherein the pH is adjusted to about 4.2.
- 10. A method of identifying a phosphorylatable substrate for a kinase enzyme, comprising:
providing a phage display peptide library wherein each peptide in the library comprises a conserved phosphorylatable amino acid residue; reacting the phage display library with the kinase and ATPγS; contacting the phage display library with a biotin HPDP; immobilizing any biotinylated phage in presence of a protein on a solid support; isolating and sequencing DNA from any phage immobilized on the solid support; and determining a phosphorylatable peptide sequence from a sequence of the DNA isolated from the phage.
- 11. A method of assaying an enzyme mediated coupling reaction, comprising:
reacting a first reactant, said first reactant comprising a fluorescent label, with a second reactant, said second reactant comprising a thiol derivative to yield a first product, said first product comprising the thiol derivative; reacting a third reactant comprising a biotin derivative with said first product at a first pH to yield a second product; adding a fourth reactant, said fourth reactant comprising a protein, to the second product; and measuring a difference in fluorescence polarization level from the second product as compared to the fluorescence polarization of the first reactant.
- 12. The method of claim 11, wherein the fluorescent label comprises a pH sensitive label.
- 13. The method of claim 11, wherein the fluorescent label comprises a pH insensitive label.
- 14. The method of claim 11, wherein the protein is selected from a group consisting of streptavidin, neutravidin and avidin.
- 15. The method of claim 1, wherein the second reactant comprises a thiophosphorylated reactant, and the enzyme comprises a kinase.
- 16. The method of claim 1, wherein the contacting step is carried out in at least a first channel of a microfluidic device.
- 17. The method of claim 11, wherein the contacting step is carried out in at least a first channel of a microfluidic device.
- 18. The method of claim 1 wherein the first reactant comprises a fluorescently labeled phosphorylatable peptide substrate.
- 19. The method of claim 1 wherein the first reactant comprises glutathione.
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of U.S. patent application Ser. No. 09/408,884, filed Sep. 29, 1999, which claims priority to Provisional U.S. Patent Application No. 60/102,486, filed Sep. 30, 1998, each of which is incorporated herein by reference in its entirety for all purposes.
Provisional Applications (1)
|
Number |
Date |
Country |
|
60102486 |
Sep 1998 |
US |
Continuation in Parts (1)
|
Number |
Date |
Country |
Parent |
09408884 |
Sep 1999 |
US |
Child |
10183040 |
Jun 2002 |
US |