Isoelectric focusing in immobilized pH gradients

Abstract

Summary The state of the art in the new technique of isoelectric focusing (IEF) in immobilized pH gradients (IPG) is extensively reviewed. The first section details the theory of the formation of narrow and ultra-narrow pH gradients on the tandem principle (one buffer, one titrant) as well as the computer program and algorithms for the generation of extended pH intervals (2 pH units). The second section is entirely devoted to the methodological aspects of IPGs: from gel casting, to polymerization kinetics and to the use of additives and pH plateaux for salt removal. The third section deals in depth with preparative aspects of IPGs, especially with regard to the optimization of environmental parameters (ionic strength, gel thickness and width of the pH gradient interval). Methods of protein detection, electrophoretic retrieval into hydroxyapatite beads, elution from Ultrogel grains and protein load as a function of polyacrylamide gel concentration are critically evaluated. The review ends with a section on the merits and limits of IPGs, possible sources of artefacts, their interpretation and possible future uses of the IPG technique.