Separation of proteins according to their isoelectric point can be performed in a pH gradient formed by stationary electrolysis of carrier ampholytes. The pH gradient is stabilized by the use of polyacrylamide, agarose, and Sephadex gels. Separated proteins can be detected by fixation with trichloroacetic acid followed by nonspecific staining, by specific staining, or through immunodiffusion techniques. Isoelectric focusing of proteins in gel media can be carried out in gel columns or on thin-layer plates by using conventional electrophoresis apparatus. Electrofocusing can be followed by electrophoresis in gel media for more complete separation of components.