Since 1980, FP immunoassays have been used extensively in the clinical laboratory to measure the concentration of drugs, both therapeutic and illicit, in biological fluids. Recently, LJL Biosystems, PanVera, and other companies have adapted the FP technique to the HTS environment. FP theory states that when a molecule is excited with plane-polarized light of the correct wavelength, it will fluoresce in the same plane after its characteristic emission lifetime, which is typically a few nanoseconds. During this time, the molecule will have tumbled randomly with respect to the original plane of excitation. If the molecule tumbles quickly with respect to the fluorescence lifetime, the fluorescence will be depolarized.
Small molecules tumble rapidly, large molecules tumble slowly.
If a small fluorescent molecule is bound to a large molecule, however, then it will tumble slowly. By measuring the extent of fluorescence polarization, this method can determine binding equilibrium and the competition for binding at a site. For example, suppose a company wants to screen for compounds that are enzyme inhibitors and that bind strongly to an active site. The FP assay would use a fluorescent marker that also binds to that enzyme's active site. Stronger inhibitors would compete more successfully for the binding sites, thus releasing more of the fluorescent marker into the unbound state.