Reproducible techniques for the prefractionation of proteins prior to two-dimensional gel electrophoresis (2-DE) are essential for increasing the number of unique proteins that can be identified and assayed following biological experimentation. A simple and robust technique for separating highly soluble (hydrophilic) cytoplasmic proteins from poorly soluble (hydrophobic) membrane-associated proteins uses differential solubility in a progressive series of extraction buffers, each containing more potent solubilizing chaotropes and detergents. This “sequential extraction” procedure is based on protein solubility in Tris buffer for the initial removal of highly soluble proteins, whereas proteins from the insoluble pellet are then extracted in 2-DE sample buffers containing urea and CHAPS. The final step of the procedure uses thiourea and amidosulfobetaine-14 (ASB-14) to solubilize CHAPS-insoluble proteins. This procedure has been optimized for the analysis of outer membrane porins from Gram negative bacteria, as well as the separation of plasma membrane proteins from mammalian cells grown in culture, and finally for the removal of insoluble cytoskeletal structures from mammalian heart tissue.