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Methods for conducting assays for enzyme activity on protein microarrays

a technology of enzyme activity and microarrays, applied in the field of methods of conducting enzyme activity assays on microarrays, can solve the problems of time-consuming process, insufficient correlation of transcriptional profiles with cellular protein levels or protein activities, and detailed analysis of individual protein biochemical properties

Inactive Publication Date: 2004-12-09
PROTOMETRIX
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

0011] The present invention provides protein chips and methods useful for the study of protein activities in a high-throughput manner. The present invention also provides methods for identifying substrates of enzymes and modulators of enzymatic activities. The invention is directed to methods of using protein chips to assay the presence, amount, functionality, activity and sensitivity to modulators of enzymes. In particular, the invention is directed to methods of conducting assays for enzymatic activity on protein microarrays. In certain embodiments, a method of the invention for assaying an enzymatic reaction comprises the following steps: (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.

Problems solved by technology

Although these studies are useful, transcriptional profiles do not necessarily correlate well with cellular protein levels or protein activities.
This is a very time-consuming process since it can take years to purify and identify a protein based on its biochemical activity.
However, a detailed analysis of an individual proteins' biochemical properties, such as, substrate specificity, kinetic profile and sensitivities to inhibitors, is a time-consuming process.
However, the size (at least 12.8 cm.times.8.6 cm) of these plates makes them unsuitable for the large-scale analysis of proteins.
Specifically, random expression libraries are tedious to screen, and contain clones that are often not full-length.
The pooling strategy obscures the actual number of proteins screened, however, and the strategy is cumbersome when large numbers of positives are identified.

Method used

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  • Methods for conducting assays for enzyme activity on protein microarrays
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  • Methods for conducting assays for enzyme activity on protein microarrays

Examples

Experimental program
Comparison scheme
Effect test

example i

6.1. Example I

Kinase Activity Assay on Microarray Materials & Reagents

[0283]

3 Materials / Equipment / Reagents Vendor Part Number Disposables / Reagents Gamma-AT.sup.33P (10 .mu.Ci / .mu.l, Perkin Elmer NEG602H250UC 250 .mu.Ci) Histone, Calf Thymus Calbiochem 38205 Casein Sigma C-4032 Myelin Basic Protein Sigma M-1891 Poly-glutamic acid-tyrosine Sigma P-2075 PBS Tablets American AB11108 Bioanalytical Tween-20 American AB02038 Bioanalytical 60 .times. 24 mm Hybridization Cover Schleicher & 10 484 907 slips Schuell Equipment Cyclone Phospho-imager Perkin Elmer B431220 8 .times. 10 Autoradiography Cassettes Fisher FB-XC-810 Phosphor Storage Screens (MS) Perkin Elmer 7001723 Lab Rotator Lab-Line 1314 Instruments Eppendorf Centrifuge (5810) Fisher Scientific 05-400-60

[0284] Reagent / Stock Preparation

[0285] Kinase Substrate Stocks

[0286] Dissolve protein substrates in 20 mM Tris to a final concentration of 10 mg / mL.

[0287] 1 L of 1.times.PBS

[0288] Dissolve 5 PBS tablets in 1 L dH2O.

[0289] Mix thorou...

example ii

6.2. Example II

Inhibitor Specificity Profiling

[0315] Fifty different kinases were immobilized on a slide together with a substrate as described in section 6.1. A mixture of Myelin Basic Protein (MBP), histone and casein was used as substrate. The kinase reactions were performed in the presence of H89 inhibitor, Rottlerin inhibitor or PP2 inhibitor (FIG. 3). The inhibitors were obtained from Calbiochem. The PP2 inhibitor is an inhibitor of tyrosine kinases. The concentration of inhibitor was 100 .mu.m for each inhibitor. The control reaction was performed in the absence of inhibitor. The specificity of the assay is demonstrated by the fact that PP2 inhibitor strongly inhibits tyrosine kinases (see circled data points in FIG. 3).

example iii

6.3. Example III

Dose-Response Analyses

[0316] Microarrays were prepared with 10 wells / slide, wherein the kinases EPHB3, FYN, and PRKCD and their substrate were immobilized in each well. The slide was coated with substrate essentially as described in section 6.1. Subsequently, a gasket with 10 openings was applied to the surface of the slide thereby creating 10 wells, i.e., the gasket provides the barriers between the wells. The accession numbers for the different kinases in the NCBI database are: for FYN: NM.sub.--002037; for PRKCD: NM.sub.--006254; and for EPHB3: NM.sub.--004443. A mixture of Myelin Basic Protein (MBP), histone and casein was used as substrate. The kinase reaction was performed in each well with a different concentration of PP2 inhibitor. The dose-response curve is shown in FIG. 4a. The data show that PP2 strongly inhibits the tyrosine kinases FYN and EPHB3 but not the serine / threonine kinase PRKCD. In a second exeriment, the kinase reaction was performed in each we...

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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.

Description

1. FIELD OF THE INVENTION[0001] 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 enzymat...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12Q1/00
CPCC12Q1/00
Inventor ZHOU, FANG X.SCHWEITZER, BARRY
Owner PROTOMETRIX
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