High-resolution protein separation procedures are an important prerequisite for proteome analyses. Classically, protein separations are based on 2D IEF/SDS-PAGE. Unfortunately, this technique only poorly recovers hydrophobic proteins, and it is not compatible with analyses of proteins in native state. Blue-native PAGE represents a powerful alternative. It is based on the careful integration of negative charge into proteins and protein complexes by the anionic wool dye Coomassie blue, and it allows protein analyses under native ("blue-native") conditions. Upon combination with SDS-PAGE for a second gel dimension, protein complexes separated by the blue-native dimension are dissected into their subunits, which form vertical rows of spots on the resulting 2D gels. 2D blue-native/SDS-PAGE ideally complements 2D IEF/SDS-PAGE in proteomics.