Two-dimensional gel electrophoresis (2-DE) is a key technique for proteomics. To analyze the proteome of PC12 cells and rat central nerve tissues, including brain and spinal cord, 2-DE technique is established. Due to much lipid and other non-protein interfering constituents, protein extraction is much more difficult for nerve tissues than for other tissues. Two different methods, i.e., precipitation with tricholoroacetic acid/acetone and ultra-centrifugation were employed to extract protein from rat brain and spinal cord for 2-DE. Other factors such as method and volume of loading sample, choice of IPG gels, concentration of SDS gels, preset of electric parameters, protocol for staining and drying the gels were also improved. Using the proper method described above, satisfactory 2-DE maps of PC12 cells, rat brain and spinal cord were obtained.