Miniaturization of fluorescence polarization receptor-binding assays using CyDye-labeled ligands.
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 approximately 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.
Harris
Alison
A
Amersham Biosciences, UK Limited, The Maynard Centre, Buckinghamshire, UK.
Cox
Sarah
S
Burns
Dougal
D
Norey
Christopher
C
D000818:Animals; D001285:Atropine / Q000378:metabolism; D001667:Binding, Competitive; D016466:CHO Cells; D002232:Carbocyanines; D006224:Cricetinae; D005454:Fluorescence Polarization / Q000379:methods; D005456:Fluorescent Dyes; D006801:Humans; D007700:Kinetics; D008904:Miniaturization; D015288:Neurokinin A / Q000378:metabolism; D010455:Peptides / Q000378:metabolism*; D010890:Pirenzepine / Q000031:analogs & derivatives* / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000378:metabolism*; D011976:Receptors, Muscarinic / Q000378:metabolism; D018040:Receptors, Neurokinin-1 / Q000378:metabolism; D012680:Sensitivity and Specificity; D013373:Substance P / Q000378:metabolism
D002232:Carbocyanines; D005456:Fluorescent Dyes; D010455:Peptides; D011956:Receptors, Cell Surface; D011976:Receptors, Muscarinic; D018040:Receptors, Neurokinin-1; C089187:cyanine dye 3; C046338:telenzepine; D013373:Substance P; D010890:Pirenzepine; D001285:Atropine; D015288:Neurokinin A