Two-dimensional gel electrophoresis (2-DE) is used to compare the protein profiles of different crude biological samples. Narrow pH range Immobilized pH Gradient (IPG) strips were designed to increase the resolution of these separations. To take full advantage of IPG strips, the ideal sample should be composed primarily of proteins that have isoelectric point (pl) values within the pH range of the IPG strip. Prefractionation of cell lysates from a human prostate cancer cell line cultured in the presence or absence of epigallocatechin-3-gallate was achieved in fewer than 30 minutes using an anion exchange resin and two expressly-designed buffers. The procedure was carried out in a centrifuge tube and standard instrumentation was used. The cell lysates were prefractionated into two fractions: proteins with pl values above 7 and between 4 and 7, respectively. The fractions were then analyzed by 2-DE, selecting appropriate pH ranges for the IPG strips, and the gels were compared with those of unprefractionated cell lysates. Protein loading capacity was optimized and resolution and visualization of the less abundant and differentially-expressed proteins were greatly improved.; Furthermore, this technique was applied successfully to eliminate albumin from serum samples. Elimination of albumin, constituting more than 50% of total serum proteins, allowed for increasing protein loads on polyacrylamide gels. Visualization and resolution of lower-abundance proteins that were previously masked by albumin was achieved as well.
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