Immobilized pH Gradients: Analytical and Preparative Use

High-resolution two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) has provided the means for detailed analysis of polypeptide constituents of whole cells or subcellular fractions from a wide range of sources. To date, for the most part 2-D PAGE has been utilized for analytical separations, following the method originally described by O'Farrell (1). Urea, reducing agent, and a nonionic detergent are used to solubilize and dissociate proteins into polypeptide subunits prior to the first-dimension isoelectric separation, using carrier ampholytes (CA). SDS-PAGE is used for the second-dimension separation based on molecular weight. 2-D PAGE has also been used successfully as the final purification step for the structural characterization of a relatively small number of polypeptides among the several hundred that can be observed for any given cell type, using analytical 2-D PAGE. For most polypeptides, structural studies are hampered in part because of the loss of resolution at higher protein loading in CA-based 2-D separations. As an alternative to the use of carrier ampholytes for 2-D PAGE, we have utilized immobilized pH gradients (IPG) for the first-dimension separation (2, 3). Derivatives of acrylamide having carboxyl or tertiary amino groups with specific pK values are used for the preparation of gels in which the pH gradient is an integral part of the polyacrylamide matrix. Precise pH ranges that have narrow or broad pH gradients may be selected (Figs. 1 and 2). The problem of cathodic drift encountered with CA is eliminated, allowing reproducible focusing of polypeptides