Our protein extraction protocol for two-dimensional gel electrophoresis (2DE) was updated to meet current needs in the field of proteomics. This protocol summarizes our experience using this method since its introduction over 30 years ago. We provide a total as well as fractionated extraction protocol. The former is easy and fast to use, suitable for most standard 2DE applications, whereas the latter is used for special applications such as the extraction of membrane or nuclear proteins. Both extraction protocols stress the need that protease inhibitors are added early to still deep frozen tissue to preclude an activation of proteases which destroy proteins and make them inaccessible to analysis. We also emphasize that, to remain soluble, proteins need to stay in an environment resembling a living cell as closely as possible. Sample dilution is therefore kept to a minimum and the pH of the extract is close to in vivo conditions at pH 7.1. In addition there are no precipitation/resolubilization steps which could irreversibly remove proteins from the extract. Furthermore, the total extraction does not even require centrifugation. Our extraction protocol is compatible with recent advances in 2DE-staining techniques such as differential in gel electrophoresis and fluorescence staining as well as mass spectrometry.