Mobility of Adsorbed Proteins Studied by Fluorescence Recovery after Photobleaching

The mobility of proteins adsorbed on a solid substrate and on a lipid monolayer was measured by fluorescence recovery after photobleaching. Both a conventional fluorescence microscope with a chargecoupled device camera and a laser scanning confocal microscope were used. For proteins adsorbed on a solid substrate, the bleached area never recovered fully, while for proteins adsorbed at liquid interfaces, the recovery was very fast. The data analysis to evaluate diffusion coefficients was done on the basis of the full intensity profiles instead of the conventional method based on recovery curves in terms of average or total intensities. The diffusion coefficients were obtained by fitting the experimental data to the intensity profiles calculated based on a solution of the diffusion equation in two dimensions, thereby reducing the possible effects of artifacts in the intensity measurements. The diffusion coefficients at the liquid interface obtained by this method were on the order of 10-7 cm²/s. Comparison with the results for proteins adsorbed on a supported lipid monolayer show that the fast recovery is related to the tangential mobility of the floating lipid monolayer.