A new two-dimensional gel technique has been developed that makes it possible to load large amounts of protein and still retain good resolution. Protein samples are added to the isoelectric-focusing acrylamide solution prior to polymerization. Focusing times are short, making it possible to complete both dimensions of a two-dimensional gel in five to six hours. The technique yields larger amounts of separated protein for sequencing, and also greatly increases the quality of two-dimensional immunoblots.