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Proceedings Paper

Robust versatile tyrosine kinase assay for HTS in drug discovery
Author(s): Sudhir S. Deshpande; I. Mineyev; John C. Owicki
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Paper Abstract

A fluorescence polarization assay was developed as an alternative to the radiolabeled SPA assays currently used to monitor the activity of tyrosine kinases in drug discovery. The assay can be used with enzymes having substrate specificity similar to that of the insulin receptor, the EGF receptor and the Src kinase receptor enzymes. The assay is easy to configure in 96, 384 and 1536-well microplates in assay volumes ranging from (mu) L with minimal efforts. The reconstituted reagents are stable for up to 24 hr at ambient temperatures, thereby minimizing the need for replenishing the stock solutions during the course of a high-throughput screen. Because of the stability and equilibrium kinetics, the assay allows the user the luxury of scheduling the reading of plates any time up to 24 hr after the completion of the assay without substantial deterioration in the assay signal. The antibody and the tracer solutions can also be premixed and added as a preformed complex in a single step. The performance of the assay with the insulin receptor kinase is described. In addition, given the diversity of the substrates used in measuring the activity of different tyrosine kinases, LJL's on-going efforts to provide different antibodies of wide ranging specificity and sensitivity are described.

Paper Details

Date Published: 21 April 1999
PDF: 11 pages
Proc. SPIE 3603, Systems and Technologies for Clinical Diagnostics and Drug Discovery II, (21 April 1999); doi: 10.1117/12.346748
Show Author Affiliations
Sudhir S. Deshpande, LJL BioSystems, Inc. (United States)
I. Mineyev, LJL BioSystems, Inc. (United States)
John C. Owicki, LJL BioSystems, Inc. (United States)


Published in SPIE Proceedings Vol. 3603:
Systems and Technologies for Clinical Diagnostics and Drug Discovery II
Gerald E. Cohn; John C. Owicki, Editor(s)

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