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The original protocol of two-dimensional electrophoresis with immobilized pH gradient (IPG-Dalt; Gorg et al., Electrophoresis 1988, 9, 531-546) is updated. Merits and limits of different methods for sample solubilization, sample application (by cup-loading or ingel rehydration) with respect to the pH interval used for IPG-isoelectric focusing are critically discussed. Guidelines for running conditions of analytical and micropreparative IPG-Dalt, using wide IPGs up to pH 12 for overview patterns, or narrow IPGs for zoom-in gels for optimum resolution and detection of minor components, are stated. Results with extended separation distances as well as automated procedures are demonstrated, and a comparison between protein detection by silver staining and fluorescent dyes is given. A brief trouble shooting guide is also included.

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Basic proteins normally lost by the cathodic drift of carrier ampholyte focusing, or separated by NEPHGE with limited reproducibility, could be well separated by two-dimensional (2-D) electrophoresis under equilibrium conditions using immobilized pH gradients (IPGs) 4-10 and 6-10 using a previously published protocol (Görg et al., Electrophoresis 1988, 9, 531-546). In the present study we have extended the pH gradient to pH 12 with IPGs 8-12, 9-12 and 10-12 for the analysis of very basic proteins. Different optimization steps with respect to pH engineering, gel composition and running conditions, such as substitution of acrylamide by dimethylacrylamide and addition of isopropanol with and without methylcellulose to the IPG rehydration solution (in order to suppress the reverse electroosmotic flow) were necessary to obtain highly reproducible 2-D patterns of ribosomal proteins from HeLa cells and mouse liver. Histones from chicken erythrocyte nuclei as well as total cell extracts of erythrocytes were also successfully separated under steady-state conditions. Due to the selectivity of isoelectric focusing in IPG 9-12, where the more acidic proteins abandon the gel, the tedious procedure of nuclei preparation prior to histone extraction can be omitted.

After having established the basic protocol of two-dimensional electrophoresis with immobilized pH gradients in the first dimension (IPG-Dalt) in 1988 (A. Görg et al., Electrophoresis 1988, 9, 531-546), some critical parameters of the actual IPG-Dalt protocols as well as the results obtained with horizontal and vertical second-dimensional sodium dodecyl sulfate-electrophoresis are demonstrated and discussed.