Isoelectric focusing in immobilized pH gradients: an update.
The latest trends on isoelectric focusing (IEF) in immobilized pH gradients (IPG) are here reviewed. The major advances on IPG technologies have been made when interfacing this technique with sodium dodecyl sulfate-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 reproducibility of spot position. With IPGs, correlation between experimental and 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 and optimization of linear, concave and convex exponential gradients, even in very complex recipes utilizing all ten Immobiline chemicals. It has also been proven that IPGs can be interfaced with mass spectrometry, 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 exploited to charter forbidden grounds, with the creation of non-linear pH gradients covering the extreme alkaline pH 10-12 gradient. In such basic regions, excellent steady-state patterns of histones and subtilisin mutants have been reported. Different families of histones could be mapped not only in this pH 10-12 interval, but also in 2-D maps exploiting this very alkaline gradient in the first dimension. Although the IPG technique is now a trouble-free, user-friendly technique, some annoying artefacts, producing severe protein smears and precipitation, were very recently reported, but found to be linked to some commercial Immobiline preparations containing up to 5% oligomers. Better quality control on the part of the company producing such chemicals should eliminate even this last source of troubles.
Righetti
P G
PG
Department of Agricultural and Industrial Biotechnologies, University of Verona, Italy.
Bossi
A
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D003198:Computer Simulation; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D007091:Image Processing, Computer-Assisted; D007525:Isoelectric Focusing / Q000379:methods*; D013058:Mass Spectrometry; D008956:Models, Chemical