Fluorescence is a technology that is now used routinely in life science research. Fluorescence reagents are used extensively to trace the presence of biomolecules in cells and other biological systems. The great advancement of fluorescence reagents has promoted a host of more complex fluorescence technologies such as fluorescence resonance energy transfer (FRET).
Fluorescence resonance energy transfer (FRET) is transfer of the excited state energy from the initially excited donor (D) to an acceptor (A). The donor molecules typically emit at shorter wavelengths that overlap with the absorption of acceptors. The process is a distance-dependent interaction between the electronic excited states of two molecules without emission of a photon. FRET is the result of long-range dipole-dipole interactions between the donor and acceptor. Excited donor molecule has several routes to release its captured energy returning to the ground state. The excited state energy can be dissipated to the environment (as light or heat) or transferred directly to a second acceptor molecule, sending the acceptor to an excited state. The latter process is called FRET.
Fluorescein isothiocyanate (FITC) is an activated precursor used for fluorescein labeling. For efficient N-terminal labeling, a seven-atom aminohexanoyl spacer
(NH2-CH2-CH2-CH2-CH2-CH2-COOH) is inserted between the fluorophore (fluoroscein) and the
N-terminus of the peptide. This spacer helps to separate the fluorophore from its point of
attachment, potentially reducing the interaction of the fluorophore with the biomolecule to which it is conjugated and making it more accessible to secondary detection reagents.