Isoelectric focusing of proteins and peptides in gel slabs and in capillaries

Abstract

The present review offers a survey of modern isoelectric focusing (IEF) techniques, including the conventional one in soluble amphoteric buffers (CA-IEF) and immobilized pH gradients (IPG). In the case of CA-IEF, recent advances on its use as a first dimension for two-dimensional electrophoresis are covered. The properties of isoelectric buffers, in terms of buffering power and conductivity, are reviewed in view of their importance as background electrolytes in capillary zone electrophoresis (CZE). In the case of IPGs, examples are given of the high correlation between theoretically predicted and experimentally measured pI values. New aspects of IPGs, such as focusing in extremely alkaline pH intervals, and its interfacing with mass spectrometry, are described. In the case of capillary IEF, new aspects of the technique are described, in particular: (a) how to modulate the slope of the pH gradient for increasing resolution; and (b) how to keep proteins in solution at, and in the proximity of, the pI value. The review ends with an excursus on the use of isoelectric buffers in capillary zone electrophoretic separations. Such buffers offer unique advantages: they permit very high voltage gradients (up to 1000 V/cm) and, thus, minimize analysis times (down to a few minutes in 30–35 cm long capillaries). This results in a marked increment in resolution due to minimal diffusion-driven peak spreading. Such buffers are finding unique applications for generating peptide maps of tryptic digests of proteins and also in the analysis of intact proteins.