Fluorescence Polarisation (FP)
Fluorescence polarisation (FP) is ideal to measure the binding of a small molecule to a much larger one. Unbound fluorescent tracers will depolarise light during emission resulting in low polarisation, because the unbound molecules tumble more rapidly than bound tracers. In contrast, bound fluorescent labelled molecules keep the polarisation orientation as their rotation is inhibited.
To evaluate the polarization two emission measurements are needed: the first using a polarized emission filter parallel to the excitation filter (S-plane) and the second with a polarized emission filter perpendicular to the excitation filter (P-plane). Typical labels are FITC, BODIPY or TMR (TRITC).
- CAMP assays
- Kinase assays
- Receptor-ligand binding