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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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The rising number of proteome projects leads to new challenges for two-dimensional electrophoresis with immobilized pH gradients and different applications of this technique. Not only wide pH gradients such as 4-12 or 3-12 (Görg et al., Electrophoresis 1999, 20, 712-717) which can give an overview of the total protein expressions of cells are in demand but also overlapping narrow immobilized pH gradients are to be used for more specialized and detailed research and micropreparative separations. The advantage of overlapping narrow pH gradients is the gain in higher resolution by stretching the protein pattern in the first dimension. This simplifies computer-aided image analysis and protein identification (e.g., by mass spectrometry). In this study the protein patterns of yeast cells in pH gradients 4-5, 4.5-5.5, 5-6, 5.5-6.7 and 6-9 are presented and compared to the pH 4-7 and 3-10 gradients. This combination allowed us to reveal a total of 2286 yeast protein spots compared to 755 protein spots in the pH 3-10 gradient.

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