Department of Pharmacology - Cambridge University, UK

Using the PHERAstar in Fluorescence Polarization mode to enhance their research.

Ca2+ is the simplest of all intracellular messengers, yet it is effectively used in all cells to regulate almost everything they do. The key to this remarkable versatility is the spatiotemporal organization of intracellular Ca2+ signals. Colin Taylor, Ana Rossi and their colleagues in the Department of Pharmacology, Cambridge are using a variety of techniques to explore the behavior of the most widely expressed of all Ca2+ channels, IP3 receptors, aiming to understand how their activation generates a hierarchy of progressively larger intracellular Ca2+ signals. The first step in this sequence is binding of IP3 to its receptor. Using a bacterially expressed N-terminal fragment of the IP3 receptor that forms the IP3-binding site, they recently developed a versatile assay that uses fluorescence polarization and the PHERAstar (BMG LABTECH) to quantify in medium-throughput assays the ligand recognition properties of this IP3-binding site.

Another great reference for the use of fluorescence polarization can be found in the ariticle "Analysis of protein-ligand interactions by fluorescence polarization" by Ana Rossi and Colin Taylor (Nature Protocols (2011)6, 365–387.

Abstract:
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 (ΔG°), enthalpy (ΔH°) and entropy (ΔS°) 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 ΔH° and ΔS°. 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. For more information or a copy of this article click here:
http://www.nature.com/nprot/journal/v6/n3/full/nprot.2011.305.html.

For more information on their work, see their website: http://www.phar.cam.ac.uk/ri/taylor.html