Protein copy number is distributed from 7 to 8 orders of magnitude in cells and probably up to 12 orders of magnitude in plasma. Classical silver-stained two-dimensional electrophoresis (2-DE) can only display up to four orders of magnitude. This is a major drawback since it is assumed that most of the regulatory proteins are low-abundance gene products. It is thus clear that the separation of low copy number proteins in amounts sufficient for postseparation analysis is an important issue in proteome studies to complete the comprehensive description of the proteome of any given cell type. The visualization of a polypeptide on a 2-DE gel will depend on the copy number, on the quantity loaded onto the gel and on the method of detection. As the amount of protein that can be loaded onto a gel is limited, one efficient solution is to fractionate the sample prior to 2-DE analysis. Several approaches exist including subcellular fractionation, affinity purification and chromatographic and electrophoretic protein prefractionation. The chromatographic step adds a new dimension in the protein separation using specific protein properties. It allows proteins to be adsorbed to a surface and eluted differentially under certain conditions. This review article presents studies combining chromatography-based methods to 2-DE analysis and draws general conclusions on this strategy.