Methods for conducting assays for enzyme activity on protein microarrays

• US 20050118665A1
• Filed: 06/09/2004
• Published: 06/02/2005
• Est. Priority Date: 06/09/2003
• Status: Active Grant

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Zhou, Fang X., Schweitzer, Barry
• Granted Patent: US 7,635,572 B2
• Time in Patent Office: Days
• US Class Current: 435/23
• CPC Class Codes: C12Q 1/00 Measuring or testing processes involving enzymes, nucleic acids or microorganisms measuring or testing apparatus with condition measuring or sensing means, e.g. colony counters, C12M1/34; Compositions therefor; Processes of preparing such compositions

Abstract

The present invention relates to methods of conducting assays for enzymatic activity on microarrays useful for the large-scale study of protein function, screening assays, and high-throughput analysis of enzymatic reactions. The invention relates to methods of using protein chips to assay the presence, amount, activity and/or function of enzymes present in a protein sample on a protein chip. In particular, the methods of the invention relate to conducting enzymatic assays using a microarray wherein a protein and a substance are immobilized on the surface of a solid support and wherein the protein and the substance are in proximity to each other sufficient for the occurrence of an enzymatic reaction between the substance and the protein. The invention also relates to microarrays that have an enzyme and a substrate immobilized on their surface wherein the enzyme and the substrate are in proximity to each other sufficient for the occurrence of an enzymatic reaction between the enzyme and the substrate.

Claims

1. A method for assaying an enzymatic reaction, the method comprising:

(a) incubating at least one protein and at least one substance under conditions conducive to the occurrence of an enzymatic reaction between the protein and the substance, wherein (i) the protein and the substance are immobilized on the surface of a solid support;

(ii) the protein and the substance are in proximity sufficient for the occurrence of said enzymatic reaction; and

(iii) the protein and the substance are not identical; and

(b) determining whether said enzymatic reaction occurs.

2. The method of claim 1, wherein the protein and the substance are immobilized before the incubating step.

3. The method of claim 1, wherein the protein, the substance, or the protein and the substance, are purified.

4. The method of claim 1, wherein the substance is a mixture of different substances.

5. The method of claim 1, wherein the method comprises immobilizing a protein and a substance on the surface of the solid support.

6. The method of claim 5, wherein the protein is immobilized prior to immobilizing the substance.

7. The method of claim 5, wherein the substance is immobilized prior to immobilizing the protein.

8. The method of claim 5, wherein the substance and the protein are immobilized simultaneously.

9. The method of claim 1, wherein the substance is a known substrate for the type of enzymatic activity assayed in said enzymatic reaction, and said determining step determines whether said protein is an enzyme having said type of enzymatic activity.

10. The method of claim 9, wherein the protein comprises a region that is homologous to the catalytic domain of an enzyme that is known to have the type of enzymatic activity assayed in said enzymatic reaction.

11. The method of claim 1, wherein the protein is an enzyme known to have the type of enzymatic activity assayed in said enzymatic reaction, and said determining step determines whether said substance is a substrate for said type of enzymatic activity.

12. The method of claim 11, wherein the enzyme is an oxidoreductase, a transferase, a hydrolase, a lyase, an isomerase, or a ligase.

13. The method of claim 12, wherein the enzyme is a kinase.

14. The method of claim 1, wherein (i) the substance is a known substrate for the type of enzymatic activity assayed in said enzymatic reaction, or a mixture of known substrates for the type of enzymatic activity assayed;

and (ii) the protein is known to catalyze the type of enzymatic activity assayed in said enzymatic reaction.

15. The method of claim 14, wherein said incubating step is done in the presence of one or more test molecules so as to determine whether said test molecules modulate said enzymatic reaction;

and said determining step comprises detecting whether a change in the amount of said enzymatic reaction occurs relative to the amount of said enzymatic reaction in the absence of the test molecules.

16. The method of claim 15, wherein said determining step comprises detecting a decrease in the rate of said enzymatic reaction relative to the rate of said enzymatic reaction in the absence of the test molecules, thereby identifying the test molecules as an inhibitor of said enzymatic reaction.

17. The method of claim 15, wherein said determining step comprises detecting an increase in the rate of said enzymatic reaction relative to the rate of said enzymatic reaction in the absence of the test molecules, thereby identifying the test molecules as an activator of said enzymatic reaction.

18. The method of claim 15, wherein the substrate is a mixture of known substrates for different subclasses of enzymes having the type of enzymatic activity assayed.

19. The method of claim 1, wherein said at least one protein is one of a plurality of different proteins organized on the surface of the solid support in a positionally addressable array.

20. The method of claim 19, wherein the substance is coated onto the surface of the solid support.

21. The method of claim 19, wherein each protein of the plurality of proteins is in proximity with the substance sufficient for the occurrence of an enzymatic reaction between the protein and the substance, and said determining step comprises determining for at least a portion of said plurality of proteins whether said enzymatic reaction occurs.

22. The method of claim 1, wherein said at least one substance is one of a plurality of different substances organized on the surface of the solid support in a positionally addressable array.

23. The method of claim 22, wherein one of the protein or the substance is coated onto the surface of the solid support and the other of the protein or the substance is printed onto the surface of the solid support.

24. The method of claim 22, wherein each substance of the plurality of different substances is in proximity with the protein sufficient for the occurrence of an enzymatic reaction between the protein and the substance, and said determining step comprises determining for at least a portion of said plurality of different substances whether said enzymatic reaction occurs.

25. The method of claim 19, wherein said plurality consists of between 2 different proteins and 10,000 different proteins.

26. The method of claim 22, wherein said plurality consists of between 2 different substances and 10,000 different substances.

27. The method of claim 1, wherein (i) a first plurality of different substances are organized on the surface of the solid support in a positionally addressable array, and (ii) a second plurality of different proteins are organized on the surface of the solid support in a positionally addressable array.

28. The method of claim 27, wherein said first plurality is between 2 different substances and 10,000 different substances, and said second plurality is between 2 different proteins and 10,000 different proteins.

29. The method of claim 27, wherein the incubating is performed for each protein of the plurality of proteins and each substance of the plurality of substances, in an identical aliquot of a reaction buffer.

30. The method of claim 1, wherein the substance, the protein, or the substance and the protein, are covalently bound to the surface of the solid support.

31. The method of claim 1, wherein the substance, the protein, or the substance and the protein, are non-covalently immobilized to the surface of the solid support.

32. The method of claim 1, wherein the substance, the protein, or the substrate and the protein, are immobilized to the surface of the solid support via a linker.

33. The method of claim 1, wherein the method further comprises quantifying the enzymatic reaction.

34. The method of claim 1, wherein the solid support has at least two wells and wherein each well comprises the same one or more different proteins immobilized on the surface of the solid support within the well.

35. The method of claim 34, wherein the solid support has at least two wells and wherein each well comprises the same set of one or more different substances immobilized on the surface of the solid support within the well.

36. The method of claim 1, wherein said determining step comprises measuring a change in a detectable signal resulting from said enzymatic reaction.