Two-dimensional electrophoresis with immobilized pH gradients: current state.

Abstract

Two-dimensional (2-D) electrophoresis as it was introduced by O’Farrell in 1975 [ 11 is unsurpassed by any other technique for simultaneously resolving hundreds of polypeptides. The usefulness of this method has been documented in thousands of publications. However, 2-D electrophoresis is still a demanding procedure with respect to performance and required expertise [2-41. Because of the large number of proteins, routine comparison of multiple 2-D patterns is carried out preferably using a computer program. However, automatic comparison is difficult when, for methodological reasons, protein migration varies. Computer-assisted pattern matching programmes can be highly accurate for similar, high-quality gels, although this accuracy decreases with decreasing quality and similarity of gels so that many spots may be mismatched. Also, when a mismatch has occurred even more unrelated spots are brought together [ 51. Additionally, for establishing collective databases by evaluating results not only from one laboratory but also from different laboratories, the importance of achieving maximum reproducibility in spot positions in 2-D patterns cannot be overemphasized. In spite of methodological improvements and the availability of sophisticated instrumentation, problems of pattern reproducibility remain when using isoelectric focusing (IEF) with carrier ampholytes. Equilibrium IEF with carrier ampholytes cannot be achieved because of pH gradient instability with time. As a result of electroendosmotic effects, the pH gradient moves to the cathode (cathodic drift) and flattens in the centre (plateau phenomenon) [6, 71. Consequently, time-dependent, not stationary protein patterns are obtained. In addition, the reproducibility of pH gradient profiles is limited by batch-to-batch variability of carrier ampholyte preparations. The storage of large amounts of one batch of carrier ampholytes or the blending of new batches in order to maintain a defined pH gradient for long-term studies will help; however, this is a tedious procedure. With the introduction of immobilized pH gradients (IPG), the problems of pH gradient stability and reproducibility have been overcome