The field of proteomics requires methods that offer high sensitivity and wide dynamic range. One of the strategies used to improve the dynamic range is sample prefractionation, such as microsolution isoelectric focusing (IEF). We have modified a commercial solution IEF instrument, the Rotofor(R) to prefractionate protein mixtures by carrier ampholyte-free solution IEF The focusing chamber of the Rotofor was divided into several compartments by polyacrylamide membranes with imbedded Immobiline mixtures of specific pH values. When an electric field is applied, each protein migrates to the compartment confined by membranes with pH values flanking its isoelectric point. The approach was demonstrated for the focusing of myoglobin into a predicted compartment, as well as the separation of a complex soluble yeast protein mixture into several distinct fractions. The proteins were dissolved in water or 30% isopropanol. The method is applicable to both gel-based and solution-phase protein identification methods, without the need for further sample preparation.