High-Throughput Fluorescence Polarization Assay of Ligand Binding to IP₃ Receptors

Abstract

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.