In this work cellular bovine prion protein (PrPc) was incorporated in supported lipid membranes and its lateral diffusion was studied by single-dye tracking (SDT) and a complementary ensemble method, fluorescence recovery after photobleaching (FRAP). PrPc was purified from calf brain with its native glycosylphosphatidylinositol (GPI) anchor and reconstituted into DMPC lipid vesicles. Homogeneous spreading on solid supports over macroscopic areas was confirmed with fluorescence microscopy. FRAP results demonstrated very high mobile fractions of up to 94%, confirming that most of the GPI-anchored PrPc are freely diffusive in the fluid supported membrane matrix. Moreover, the lateral diffusivity of PrPc significantly depends on the pH of the buffer, suggesting that the conformation of PrPc and thus the frictional drag exerted to the protein molecule (and thus the effective hydrodynamic radius) is influenced by the effective net charge. To complement the ensemble results obtained by FRAP, the statistical variation of lateral diffusion coefficients of individual PrPc molecules in the supported membranes were measured with SDT. Simulation-based statistical analysis indicated that in addition to the expected statistical scatter there is a significant spread of diffusion coefficients, while the average of the diffusion coefficients of individual proteins obtained by SDT is in excellent agreement with those measured by ensemble FRAP. In further experiments, PrPc was laterally concentrated in the membrane by the application of tangential electric fields (membrane electrophoresis). However, the equilibrium concentration profile reached after 20 min was different from an exponential gradient. This finding suggests that PrPc purified from bovine brain possesses non-uniform net charges. As the lateral diffusion coefficient of proteins in two-dimensional lipid membranes sensitively depends upon the frictional drag, the combination of SDT, ensemble FRAP, and membrane electrophoresis can be used as a powerful tool to gain insights into protein-protein binding and oligomer formation that would play a crucial role in infectious protein transmitted diseases such as BSE. (C) 2007 Elsevier B.V. All rights reserved.