Recently, following the pioneering experiments of Juan Yguerabide (personal communication), a fluorescence photo-bleaching technique has been widely used to measure lateral diffusion rates in the plane of biological membranes (for reviews see refs 1 and 2). In this technique, photobleaching of fluorescently labelled probe molecules by brief exposure to an intense laser light source is used to generate localized depletions of probe molecules. Diffusion coefficients are determined from the kinetics of fluorescence redistribution after photobleaching (FRAP), monitored by an attenuated light source. Despite the widespread application of the FRAP technique, questions remain as to whether or not dye-sensitized photodamage might seriously affect the results3-6. To test for such effects, the diffusion characteristics of a membrane system should be compared with and without fluorescence photobleaching, but this is generally extremely difficult to do - an indication of the potential capabilities of the photobleaching technique. We have now devised such a test for the diffusion of integral membrane proteins in erythrocyte membranes. We report that, within experimental error, membrane photodamage does not seem to affect the results observed in this system.
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