Fluorescence polarization measurements are made using an optical system that includes polarizing filters in the light path. 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. For example, large molecules (e.g. proteins) in solution, rotate relatively slowly because of their size and will emit polarized light when excited with polarized light. The fast rotation of smaller molecules will result in a depolarization of the signal. The emission system uses polarizing filters to analyze the polarity of the emitted light. A low level of polarization indicates that small fluorescent molecules move freely in the sample. A high level of polarization indicates that fluorescent molecule is attached to a larger molecular complex. Examples of FP-based assays include molecular binding assays, since they allow the detection of a small fluorescent molecule binding (or not) to a larger, non-fluorescent molecule: binding results in a slower rotation speed of the fluorescent molecule, and in an increase in the polarization of the signal.
Molecules are excited with light. Unbound small
molecules emit depolarized light.
Small molecules bound to larger ones emit polarized light.
FP is widely used in research labs to study molecular binding or dissociation events and in screening labs to screen for drug candidates. There are many FP assay kits available for a wide variety of applications.