Fluorescence Polarization

This assay marks a simple, fast and cheap way to determine displacement data in vitro. As this assay does not require intracellular target binding, tracers can consist of small molecules of natural ligands such as DNA, RNA or peptides coupled to a fluorescent dye. The assay principle is reliant on polarized light to excite the fluorophore, while simultaneously detecting polarized and depolarized light. If an unbound tracer is in solution, the degree of depolarization correlates with its tumbling rate leading to the emission of mainly depolarized light. Binding to a protein results in an increase in the mass of the complex, which, in turn, reduces its tumbling rate and leads to an increased emission of polarised light. Therefore, the binding of the tracer to a protein can be detected by observing the increase in polarized light intensity emitted during a protein titration experiment, leading to the determination of the tracer K_D. After determining the K_D value, displacement can be measured by carrying out compound titration experiments with constant concentrations of both the tracer and protein.

As this assay is based on in vitro measurements, it is necessary to have purified protein that contains the relevant binding site. However, fluorescence polarization allows for the measurement of intercellular molecules, such as DNA, RNA, and peptides, to determine K_i. For measurement, a fluorescently labelled ligand is used in a constant concentration which is chosen based on the optimal fluorescence signal. Subsequently, the protein is titrated to the tracer for K_D determination where generally 2-fold excess displays the concentration to use in the assay setup. Therefore, the concentration recommendation for fluorescence polarization assays describes the recommended protein concentration while the tracer is used as described below the experimental data.

For FP, an important consideration is the use of the so called Nikolovska-Coleska equation¹ rather than the Cheng-Prusoff equation² for K_i determination.

Due to its low cost and ease of use, fluorescence polarization assays can be used for high-throughput applications as described here.

1. ¹ Nikolovska-Coleska, Zaneta, et al. "Development and optimization of a binding assay for the XIAP BIR3 domain using fluorescence polarization." Analytical biochemistry 332.2 (2004): 261-273.

2. ² Yung-Chi, Cheng, and William H. Prusoff. "Relationship between the inhibition constant (KI) and the concentration of inhibitor which causes 50 per cent inhibition (IC50) of an enzymatic reaction." Biochemical pharmacology 22.23 (1973): 3099-3108.