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Fluorescence Polarization Competition Assay: The Range of Resolvable Inhibitor Potency Is Limited by the Affinity of the Fluorescent Ligand

Wyeth Research Division of Biological Chemistry 401 N. Middletown Road Pearl River, NY 10965, huangx{at}wyeth.com

For the development of fluorescence polarization (FP) competition assays, there is a widespread belief that tight-binding fluorescent ligands should be avoided to identify inhibitors of low or intermediate potency in the screening of small-molecule compound libraries. It is demonstrated herein that this statement is a misconception; in fact, the higher the affinity of the fluorescent ligand, the wider the range of inhibitor potency that can be resolved. An approximate estimate for the low end of inhibitor K_i values that can be resolved is the K_d value of the fluorescent ligand. Because FP competition assays are typically conducted under nonstoichiometric titration conditions, it is suggested that a fluorescent ligand of highest affinity that also has an adequate quantum yield to satisfy such conditions be selected. (Journal of Biomolecular Screening 2003:34-38)

Key Words: Fluorescence polarization • competition assay • high-throughput screening • stoichiometric titration

Journal of Biomolecular Screening, Vol. 8, No. 1, 34-38 (2003)
DOI: 10.1177/1087057102239666


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