AbstractHorizontal two‐dimensional (2‐D) electrophoresis with immobilized pH gradients (IPG) in the first dimension, described for soluble proteins inElectrophoresis1985, 6, 599–604, has been extended to an analysis of complex protein mixtures, such as leukemia cell proteins or bean proteins, in the presence of nonionic detergents. By optimizing the ratio of gel volumes for the first and second dimensional separation, and by decreasing the concentration of Nonidet P‐40 in the IPG gel, undistorted protein patterns in the 2‐D gel are obtained. Horizontal and vertical streaking are considerably diminished by optimized reswelling conditions of the dry IPG strips in presence of both urea and detergent. Resolution is improved and a reduced background is obtained when overcrowded gel areas are spread by flattening the pH gradient (in the first dimension) and the polyacrylamide gradient (in the second d