Different fluorescence-based techniques are used to study the protein dynamics in living cells. These techniques include FRAP, FRET, and PET.
Fluorescent recovery after photobleaching (FRAP) is a fluorescent-protein-based detection technique used to quantify protein movement rates within the cell. This method exposes a small portion of the cell to an intense laser beam. The laser beam causes permanent photobleaching of the fluorophore-tagged proteins in the exposed region. As the bleached proteins diffuse out over time and fluorescently-labeled proteins from other parts of the cell move in, the region becomes fluorescent again. The rate of movement is quantified by plotting the relative fluorescence intensity versus the time taken, which allows for determining the rate with which protein moves within the cell.
Förster resonance energy transfer (FRET) is a molecular technique used to determine the distance between two proteins. Thus, FRET can be used as a molecular ruler. The technique uses two fluorophore-tagged proteins, one acting as the donor and the other as the acceptor. Upon excitation at a specified wavelength, the donor fluorophore emits fluorescence energy that is absorbed by the acceptor fluorophore. The donor fluorophore returns to the ground state upon energy transfer, while the acceptor fluorophore emits fluorescence that is visualized with a fluorescence microscope. The FRET technique depends on three factors, the distance between interacting proteins, the extent of spectral overlap between the donor and acceptor fluorophores, and the orientation of the donor and acceptor fluorophore during energy transfer. The energy transfer between the correctly oriented donor and acceptor fluorophores can only occur when the distance between the two interacting proteins is 10 nm or less.
Photoinduced Electron Transfer or PET determines the sub-atomic distance between proteins in a cell. In PET, the fluorophore absorbs the light and emits a fluorescence signal through an excited electron. The excited electron is transferred to the receptor. During the energy transfer, a redox reaction occurs, generating charge separation between the donor and acceptor proteins.
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