Two-Dimensional Electrophoresis with Immobilized pH Gradients

Abstract

Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) (O’Farrell 1975), in which proteins are separated according to charge (pI) by isoelectric focusing (IEF) in the first dimension and according to size (M r ) by SDS-PAGE in the second dimension, has a unique capacity for the resolution of complex mixtures of proteins, permiting the simultaneous analysis of hundreds or even thousands of gene products. However, the exchange of 2-D gel data between laboratories has been a major problem because of spatial irreproducibility between 2-D gels generated by the conventional method of 2-D PAGE using carrier ampholyte (CA) IER Equilibrium CA-IEF cannot be achieved because of pH gradient instability with prolonged focusing time, as the pH gradient moves towards the cathode (‘cathodic drift’) and flattens in the centre (‘plateau phenomenon’). Consequently, time-dependent protein patterns are obtained. In addition, reproducibility of pH gradient profiles is limited by the batch-to-batch variability of CA preparations.