Two-dimensional gel electrophoresis has been the technique of choice for analyzing the protein composition of cell types, tissues and fluids and is a key technology in modern proteomics projects. Here we describe reproducible procedures for running isoelectric focusing and nonequilibrium pH gradient electrophoresis gels that are based on the carrier ampholyte technology originally described by O'Farrell. Moreover, we present a sensitive immunoblotting procedure that has been used routinely in our laboratory to determine the identity of hundreds of human proteins.