ABSTRACT
Two-dimensional gel electrophoresis (2DE) is a powerful technique that resolves complex protein mixtures in the fi rst dimension by isoelectric point (pI) and in the second dimension by molecular weight. The introduction of immobilized pH gradient (IPG) strips has signifi cantly improved 2DE separation in a reproducible manner.1 However the number of the proteins that can be resolved and visualized by 2DE is limited.2 A large format gel (20 25 cm) can only portray approximately 1500 to 2000 protein spots, while a mammalian cell probably contains more than 20,000 protein species, and a single mammalian tissue may represent a mixture of more than 50,000 protein species.3 The limitations in protein detection and quantifi cation are mainly due to the huge dynamic range of cellular protein expression and the presence of poorly resolved protein smears or streaks instead of distinct protein spots in the gel. While strategies have been employed to address the issue of dynamic range, for example, the application of various sample complexity reduction techniques (subcellular fractionation, solution-phase isoelectric focusing [IEF], affi nity enrichment or depletion, and chromatographic separation) and the application of more sensitive gel staining techniques,4 efforts have also been made to reduce or eliminate protein streaks.
