Two-dimensional polyacrylamide gel electrophoresis (PAGE) is used for separation of complex protein mixtures by the independent parameters of isoelectric point and molecular weight. Isoelectric focusing (IEF) separates proteins in a pH gradient. Each protein is 'focused' because it moves under the influence of the electric field until it reaches its isoelectric point, the pH at which it has no net charge. After IEF in the presence of urea and a nonionic detergent, the IEF gel is equilibrated in sodium dodecyl sulfate (SDS) to prepare the proteins for SDS-PAGE. The method described here1 uses carrier ampholytes to form a pH gradient in a long, thin (1.2-mm) focusing gel composed of a low percentage (2.7%) of acrylamide and containing 9.5 M urea and 2% Nonidet P-40 to maintain protein solubility. After IEF, the gel is briefly equilibrated in SDS and placed directly onto the top edge of a second-dimension slab gel. Because the time between the end of IEF and the start of SDS-PAGE is only a few minutes (thereby minimizing diffusion), the spots are highly resolved and nearly round in shape.
