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Miniaturization of Fluorescence Polarization Receptor-Binding Assays Using CyDye-Labeled Ligands

Bristol, UK

Sarah Cox

Amersham Biosciences UK Limited, Buckinghamshire, UK

Dougal Burns

Amersham Biosciences UK Limited, Buckinghamshire, UK

Christopher Norey

Amersham Biosciences UK Limited, Buckinghamshire, UK

Fluorescence polarization (FP) is an established technique for the study of biological interactions and is frequently used in the high-throughput screening (HTS) of potential new drug targets. This work describes the miniaturization of FP receptor assays to 1536-well formats for use in HTS. The FP assays were initially developed in 384-well microplates using CyDye-labeled nonpeptide and peptide ligands. Receptor expression levels varied from 1 to 10 pmols receptor per mg protein, and ligand concentrations were in the 0.5- to 1.0-nM range. The FP assays were successfully miniaturized to 1536-well formats using Cy3B-labeled ligands, significantly reducing reagent consumption, particularly the receptor source, without compromising assay reliability. Z' factor values determined for the FP receptor assays in both 384- and 1536-well formats were found to be > 0.5, indicating the assays to be robust, reliable, and suitable for HTS purposes.

Key Words: fluorescence polarization • high-throughput screening • CyDye-labeled nonpeptide and peptide ligands • miniaturization of FP receptor assays

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