ISOELECTRIC-FOCUSING IN IMMOBILIZED PH GRADIENTS - AN UPDATE

The latest trends on isoelectric focusing (IEF) in immobilized pH grad ients (IPG) are here reviewed. The major advances on IPG technologies have been made when interfacing this technique with sodium dodecyl sul fate-polyacrylamide gel electrophoresis to produce two-dimensional (2- D) maps. Previous 2-D maps were routinely performed using conventional IEF as a first dimension, which typically resulted in poor reproducib ility of spot position. With IPGs, correlation between experimental an d calculated protein pi values is as good as +/-0.01 to 0.02 pH units. A new software has also been released, permitting easy calculation an d optimization of linear, concave and convex exponential gradients, ev en in very complex recipes utilizing all ten Immobiline chemicals. It has also been proven that IPGs can be interfaced with mass spectrometr y, thus obtaining a novel 2-D map with the best of pi measurements in the first dimension coupled with the best of mass determination in the second dimension. Recently, it has been shown that IPGs can be exploi ted to charter forbidden grounds, with the creation of non-linear pH g radients covering the extreme alkaline pH 10-12 gradient. In such basi c regions, excellent steady-state patterns of histones and subtilisin mutants have been reported. Different families of histones could be ma pped not only in this pH 10-12 interval, but also in 2-D maps exploiti ng this very alkaline gradient in the first dimension. Although the IP G technique is now a trouble-free, user-friendly technique, some annoy ing artefacts, producing severe protein smears and precipitation, were very recently reported, but found to be linked to some commercial Imm obiline preparations containing up to 5% oligomers. Better quality con trol on the part of the company producing such chemicals should elimin ate even this last source of troubles. (C) 1997 Elsevier Science B.V.