Analyses of Ligand Binding to IP3 Receptors Using Fluorescence Polarization.
Rossi, Ana M
Taylor, Colin W
Methods Mol Biol
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Rossi, A. M., & Taylor, C. W. (2020). Analyses of Ligand Binding to IP3 Receptors Using Fluorescence Polarization.. Methods Mol Biol, 2091 107-120. https://doi.org/10.1007/978-1-0716-0167-9_9
Fluorescence polarization (FP) can be used to measure binding of a small fluorescent ligand to a larger protein because the ligand rotates more rapidly in its free form than when bound. When excited with plane polarized light, the free fluorescent ligand emits depolarized light, which can be quantified. Upon binding, its rotation is reduced and more of the emitted light remains polarized. This allows FP to be used as a nondestructive assay of ligand binding. Here we describe a fast, high-throughput FP assay to quantify the binding of fluorescently labeled inositol 1,4,5-trisphosphate (IP3) to N-terminal fragments of the IP3 receptor. The assay is fast (1-6 h), it avoids use of radioactive materials and when measurements are performed at different temperatures, it can resolve Gibbs free energy (ΔG°), enthalpy (ΔH°), and entropy (ΔS°) changes of ligand binding.
Affinity, Fluorescence polarization, IP3 receptor, Ligand binding, Thermodynamics, Entropy, Fluorescence Polarization, Inositol 1,4,5-Trisphosphate, Inositol 1,4,5-Trisphosphate Receptors, Ligands, Thermodynamics
Biotechnology and Biological Sciences Research Council (BB/P005330/1)
Wellcome Trust (101844/Z/13/Z)
External DOI: https://doi.org/10.1007/978-1-0716-0167-9_9
This record's URL: https://www.repository.cam.ac.uk/handle/1810/288097
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