Using FRAP, one can measure the rate at which membrane lipid or protein molecules move - the diffusion coefficient, as well as the proportion of the molecules that are laterally mobile.
You take a cell and designate a specific area of the membrane that you are interested in. The entire membrane is tagged with GFP and is fluorescent. You then take a laser and photobleach the area of the membrane that you designated. At that point, t=0, the area has no fluorescence. You then measure the amount of time the fluorescence returns to that area. When fluroescence returns to the area, you know that the membrane proteins have moved laterally through the membrane and been replaced with non-photobleached membrane proteins.
Protocol
1. Cells are labeled with a fluorescent reagent that binds uniformly to a specific membrane lipid or protein
2. Laser light is focused on a small area of the surface, irreversibly bleaching the bound reagent and thus reducing the fluorescence in the illuminated area
3. In time, fluorescence of the bleached patch increases as unbleached fluorescent surface molecules diffuse into it and bleached ones diffuse outward. The extent of recovery of fluorescence in the bleached patch is proportional to the fraction of labeled molecules that are mobile in the membrane. From this information, you can calculate the diffusion coefficient of the protein or lipid.
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