Fluorescence Polarization

Fluorescence polarization detection is based on the excitation of a fluophore, similar as in standard Fluorescence Intensity (FI). However, the difference is that the optical system includes polarizing filters: the samples in the microplate are excited using polarized light and depending on the mobility of the fluorescent molecules found in the wells, the light emitted will either be polarized or not. Large molecules such as proteins in solution rotate relatively slowly because of their size, and will emit polarized light when excited with polarized light. Conversely, fast rotation of smaller molecules will result in a depolarized signal. A low level of polarization indicates that fluorescently labelled  molecules move freely in the solution. A high level of polarization then means that a larger molecular complex is present, which rotates slowly due to the size. Given that, applications of FP include binding assays or more recently the Predictor hERG assay.

BMG LABTECH has published numerous application notes (AN) on using fluorescence polarization detection mode with various BMG LABTECH microplate readers including:

• High-throughput protein-DNA affinity measurements using fluorescence anisotropy (AN 180)
• New Transcreener® ADP2 FP Assay performed on BMG LABTECH's PHERAstar Plus HTS Microplate Reader (AN 182)
• Performance of the PredictorTM hERG Fluorescence Polarization Assay Kit using the PHERAstar (AN 173)
• Membrane Fluidity Measurements Using UV Fluorescence Polarization and the POLARstar Omega (AN 205)

Click here for a complete list of fluorescence polarization application notes.

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 6, 365–387 (2011).

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 see: http://www.bmglabtech.com/customer-focus/cambridge-pharmacology.cfm

The following BMG LABTECH microplate readers can be configured to perform fluorescence polarization measurements: