Two-Dimensional Electrophoresis Using Immobilized pH Gradients in the First Dimension

Abstract

The high-resolution capacity of two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) based on the method of O’Farrell (1) makes it the method of choice for the analysis of complex protein mixtures (2–4). In the standard procedure, cylindrical tube isoelectric focusing (IEF) gels are used. Unfortunately this system suffers from severe cathodic drift resulting in pH gradients that do not extend above pH 8, resulting in concomitant loss of basic proteins from 2-D maps. Gradients can be extended to pH 10 by special treatment of the glass IEF tubes (5) or by using a horizontal flat-bed IEF (5–7). In spite of the improvement in resolution of basic proteins that can be achieved with these procedures, difficulties are still associated with the first-dimension IEF gels as a consequence of the characteristic properties of the synthetic carrier ampholytes used to generate the pH gradients. These include problems of batch reproducibility of ampholytes, irreproducibility of separations, difficulty in control of pH gradient stability and shape (i.e., “pH gradient engineering”), and the possibility of artifacts caused by protein-ampholyte interactions.