Use of IRS-polypeptides for identifying of pharmaceutically active compounds
View Patent ↗An assay for determining the ability of an enzyme, functional fragment, or functional derivative thereof to modify the phosphorylation status of a biotinylated polypeptide.
1. A method for determining the ability of a first polypeptide to modify the phosphorylation status of a second, biotinylated polypeptide, the method comprising the steps of
a. contacting the first polypeptide with the second polypeptide in a suitable reaction mixture;
b. contacting the reaction mixture with a means which is coupled to a carrier and is able to bind to the second polypeptide; and
c. determining the phosphorylation state of the second polypeptide,
wherein the carrier comprises a first carrier and a second carrier, wherein the first carrier comprises a first signal generator and the second carrier comprises a second signal generator, wherein the first carrier is coupled to the means and the second carrier is coupled to the second polypeptide.
2. The method of claim 1 , wherein the means comprises a phospho-specific antibody.
3. The method of claim 1 , wherein the means comprise streptavidin.
4. The method of claim 1 , wherein the step of determining the phosphorylation state comprises determining whether a signal has been generated.
5. The method of claim 1 , wherein the second polypeptide has a length of at least about 50 amino acids.
6. The method of claim 5 , wherein the second polypeptide has a length of about 50 to 300 amino acids.
7. The method of claim 6 , wherein the second polypeptide has a size of at least about 1 to 100 kda.
8. The method of claim 6 , wherein the second polypeptide has a size of about 10 to 50 kda.
9. The method of claim 6 , wherein the second polypeptide has a size of about 25 to 35 kda.
10. The method of claim 1 , wherein the first polypeptide is an enzyme.
11. The method of claim 10 , wherein the first polypeptide is a kinase.
12. The method of claim 11 , wherein the first polypeptide is a tyrosine kinase.
13. The method of claim 1 , wherein the first polypeptide is insulin receptor, IGF-1 receptor, trK receptor, EGF receptor, casein kinase II, protein kinase C, protein kinase B/Akt, mitogen-activated protein kinase (MAP kinase), GSK-3, ERK, JNK, or a functional fragment of any of the foregoing.
14. The method of claim 1 , wherein the first polypeptide is a functional fragment of an enzyme or a functional derivative of an enzyme.
15. The method of claim 1 , wherein the second polypeptide is a natural substrate of the first polypeptide.