Pharmacia & Upjohn Company

Charles K Marschke

Dennis E Epps

Paul K Tomich

Ferenc J Kezdy

Ronald W Sarver

The invention relates to novel fluorescence-based assays for protein kinases and phosphatases which can be used in high throughput screening. The methods of the invention utilize a competitive immunoassay to determine the amount of substrate that is phosphorylated or dephosphorylated during the course of a kinase or phosphatase reaction to yield a product, as well as the phosphorylating or dephosphorylating activity of a kinase or phosphatase.

Fluorescence-based high throughput screening assays for protein kinases and phosphatases

The present invention provides a method for identifying a test compound that binds to a target species. The method includes: incubating at least one test mixture under isothermal denaturing conditions, each test mixture comprising at least one test compound, and at least one target species, wherein the isothermal denaturing conditions are effective to cause at least a portion of the target species to denature to a measurable extent; detecting a denaturation signal of each target species in the presence of the at least one test compound by a change in the diffusion properties of the target molecule using fluorescence correlation spectroscopy; and comparing the denaturation signal of each target species in the presence of at least one test compound with a denaturation signal of the same target species in the absence of the at least one test compound under the same isothermal denaturing conditions.

Methods of identifying compounds that bind to target species under isothermal denaturing conditions

Use of fluorescence correlation spectroscopy to identify compounds that bind to target species under isothermal denaturing conditions

A high throughput screening method for identifying a compound that binds to a target species. The method includes: incubating a plurality of test mixtures under isothermal denaturing conditions, each test mixture including at least one test compound and at least one target species, wherein the isothermal denaturing conditions are effective to cause at least a portion of the target species to denature to a measurable extent. The method further involves detecting a denaturation signal of each target species in the presence of the at least one test compound; and comparing the denaturation signal of each target species in the presence of the at least one test compound with a denaturation signal of the same target species in the absence of the at least one test compound under the same isothermal denaturing conditions.

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Kalamazoo, MI, United States of America

Charles K Marschke