Using Ultra-Zoom Gels for High-Resolution Two-Dimensional Polyacrylamide Gel Electrophoresis

Abstract

Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) is one of the core Technologies—together with mass spectrometry—of proteome research. It is the only method currently available that is able to simultaneously separate the thousands of proteins found in biological samples. The method originates from the seminal work of O’Farrell and Klose in the 1970s (1,2). The main drawback of the original method was the poor reproducibility, due to the instability of the carrier ampholyte pH gradient in the first dimension. Insufficient sample loading capacity was another limitation. With the introduction of immobilized pH gradients (IPGs), the problems of gradient instability and loading capacity were largely overcome (3). The work of Görg and co-workers has further refined the technology with respect to the separation of proteins with extreme pI values (4). In short, the technology has made substantial improvements, but in many studies proteome analysis is carried out on single wide-range pH gels—for example, on pH 3.0–10.0 or on pH 4.0–7.0. While the first gradient lacks the high resolution (1500–2000 proteins per gel depending on the staining procedure) necessary to separate subtle differences between proteins, the latter does not cover the complete pH range. One possibility to obtain higher resolution and visualize more proteins is the use of a larger gel format, where up to 9000 proteins can be detected. However, this methodology makes use of carrier ampholyte-based IEF technology and large second-dimension gels. These gels are not commercially available (5).