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

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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 mgr. 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 mgr 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 agr Fluorescence Polarization Assay; Fluorescence Polarization Comparison with Prompt Fluorescence; Scintillation Proximity Assay

Language: English

Document Type: Regular paper

DOI: 10.2174/1386207003331463

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