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Cold Spring Harb Protoc. 2013 Oct 1;2013(10):938-46. doi: 10.1101/pdb.prot073080.

High-throughput fluorescence polarization assay of ligand binding to IP3 receptors.

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  • 1Department of Pharmacology, University of Cambridge, Cambridge CB2 1PD, United Kingdom.


Fluorescence polarization (FP) allows quantification of the binding of a small fluorescent ligand to a larger protein because the free ligand rotates more rapidly than the bound form. This protocol describes an FP assay for the binding of fluorescein-labeled inositol 1,4,5-trisphosphate (IP3) to amino-terminal fragments of the IP3 receptor at different temperatures and in the presence of competing ligands. The method requires fluorescein-labeled IP3 and a plate-reader capable of FP measurements. The assay can measure low-affinity interactions in real time, it avoids use of radioactive materials, is nondestructive, and can resolve changes in Gibbs free energy (ΔG°), enthalpy (ΔH°), and entropy (ΔS°) that occur with ligand binding. It is applicable to any purified protein for which a fluorescent ligand is available. After optimization, the procedure can be completed in 1-6 h.

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