Fluorescence Recovery After Photobleaching - Applications Outside The Membrane

Applications Outside The Membrane

FRAP can also be used to monitor proteins outside the membrane. After the protein of interest is made fluorescent, generally by expression as a GFP fusion protein, a confocal microscope is used to photobleach and monitor a region of the cytoplasm, mitotic spindle, nucleus, or another cellular structure. The mean fluorescence in the region can then be plotted versus time since the photobleaching, and the resulting curve can yield kinetic coefficients, such as those for the protein's binding reactions and/or the protein's diffusion coefficient in the medium where it is being monitored. Often the only dynamics considered are diffusion and binding/unbinding interactions, however, in principle proteins can also move via flow, i.e., undergo directed motion, and this was recognized very early by Axelrod et al. This could be due to flow of the cytoplasm or nucleoplasm, or transport along filaments in the cell such as microtubules by molecular motors.

The analysis is most simple when the fluorescence recovery is limited by either the rate of diffusion into the bleached area or by rate at which bleached proteins unbind from their binding sites within the bleached area, and are replaced by fluorescent protein. Let us look at these two limits, for the common case of bleaching a GFP fusion protein in a living cell.