Journal
NATURE PROTOCOLS
Volume 6, Issue 3, Pages -Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/nprot.2011.305
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Funding
- Wellcome Trust [085295]
- Biotechnology and Biological Sciences Research Council [BB/H009736]
- BBSRC [BB/H009736/1] Funding Source: UKRI
- Biotechnology and Biological Sciences Research Council [BB/H009736/1] Funding Source: researchfish
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Quantification of the associations between biomolecules is required both to predict and understand the interactions that underpin all biological activity. Fluorescence polarization ( FP) provides a nondisruptive means of measuring the association of a fluorescent ligand with a larger molecule. We describe an FP assay in which binding of fluorescein-labeled inositol 1,4,5-trisphosphate (IP3) to N-terminal fragments of IP3 receptors can be characterized at different temperatures and in competition with other ligands. The assay allows the standard Gibbs free energy (Delta G degrees), enthalpy (Delta H degrees) and entropy (Delta S degrees) changes of ligand binding to be determined. The method is applicable to any purified ligand-binding site for which an appropriate fluorescent ligand is available. FP can be used to measure low-affinity interactions in real time without the use of radioactive materials, it is nondestructive and, with appropriate care, it can resolve Delta H degrees and Delta S degrees. The first part of the protocol, protein preparation, may take several weeks, whereas the FP measurements, once they have been optimized, would normally take 1-6 h.
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