How FRAP works
In unbleached cells fluorescent molecules are in equilibrium. Bleaching a fraction of the total fluorescent molecules in a region of interest (ROI) disturbs this equilibrium. Under optimal conditions the recovery kinetics are dependent only on the mobility (effective diffusion coefficient and binding kinetics to macromolecular structures) of the investigated molecule.
Thus by measuring the recovery kinetics, following properties of the molecule can be characterized:
- the ratio between mobile and immobile fraction
- the effective diffusion coefficient Deff
- the binding time (as well as assembly/disassembly) of proteins to macromolecular structures
- continuity of intracellular organelles (FLIP)
- formation of protein complexes (resulting in lower Deff)