Fluorescence Polarization is a Useful Technology for Reagent Reduction in Assay Miniaturization

Authors: Kowski T.J.; Wu J.J.

Source: Combinatorial Chemistry & High Throughput Screening, Volume 3, Number 5, October 2000 , pp. 437-444(8)

Publisher: Bentham Science Publishers

Abstract:

The use of fluorescence polarization (FP) has increased significantly in the development of sensitive and robust assays for high throughput screening of chemical compound libraries during the past few years. In this study, we show that FP is a useful assay miniaturization technology for reagent reduction during high throughput screening. We developed and optimized several FP assays for binding to estrogen receptor ??and two protein kinases with an assay volume of 100 . Without any re-optimization, a consistent signal window was maintained in 384- or 1536-well format when the assay volume varied from 2.5-100 at constant concentrations of all assay components. In contrast, the signal window decreased with decreasing assay volume at constant reagent concentration in the protein kinase C scintillation proximity assay (SPA) and prompt fluorescence assay. In addition, the effect of evaporation on the signal window was minimal for the FP assays. Our study suggests that FP is superior to SPA and prompt fluorescence in terms of reagent reduction in the miniaturized assay format.

Keywords: Fluorescence Polarization; Reagent Reduction; Assay Miniaturization Technology; Estrogen Receptor [agr] Fluorescence Polarization Assay; Protein Kinase; Protein Kinase C (PKC) Fluorescence Polarization Assay; Protein Kinase C Scintillation Proximity Assay; Miniaturization of Estrogen Receptor Fluorescence Polarization Assay; Fluorescence Polarization Comparison with Prompt Fluorescence; Scintillation Proximity Assay

Language: English

Document Type: Regular paper

DOI: http://dx.doi.org/10.2174/1386207003331463

Publication date: 2000-10-01

• Combinatorial Chemistry & High Throughput Screening publishes full length original research articles and reviews describing various topics in combinatorial chemistry (e.g. small molecules, peptide, nucleic acid or phage display libraries) and/or high throughput screening (e.g. developmental, practical or theoretical). Ancillary subjects of key importance, such as robotics and informatics, will also be covered by the journal. In these respective subject areas, Combinatorial Chemistry & High Throughput Screening is intended to function as the most comprehensive and up-to-date medium available. The journal should be of value to individuals engaged in the process of drug discoveryand development, in the settings of industry, academia or government.

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