Isoelectric focusing in immobilized pH gradients: generation and optimization of wide pH intervals with two-chamber mixers |
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Authors: | E Gianazza G Dossi F Celentano P G Righetti |
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Institution: | 1. Chair of Biochemistry, School of Pharmaceutical Sciences and Department of Biomedical Sciences and Technologies, University of Milan, Via Celoria 2, Milan 20133, Italy;2. Cattedra di Chimica Biologica III, University of Milan, San Raffaele Institute, Via Olgettina 60, Milan 20132 Italy;3. Department of Biology, University of Milan, Via Celoria 26, Milan 20133, Italy |
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Abstract: | A new technique for generating extended pH gradients (5 pH units) in Immobiline gels is reported. The previously described (J. Biochem. Biophys. Methods 7, 1983, 123-142) five-chamber gradient mixer has been replaced by a two-vessel device. A single mixture of the available Immobilines (pK 3.6, 4.6, 6.2, 7.0, 8.5 and 9.3) is made, with relative concentrations adjusted so as to produce the most uniform buffering power throughout the desired pH interval. This mixture is then divided into two portions, which are titrated to the extremes of the required pH span with an acidic titrant (Immobiline pK approximately 1) and a basic species (Immobiline pK 9.95). Highly reproducible pH gradients (pH 4-9) are thus generated, which appear extremely useful for the first dimensioned of 2-dimensional techniques. Our previously reported computer program has been implemented with an optimization algorithm which, given any cocktail of Immobilines, automatically adjusts the relative initial concentrations until the smoothest possible beta power is found. For the first time it is possible to perform IEF under controlled physico-chemical parameters: pH span and linearity, beta power, ionic strength and molarity of the buffering species. |
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Keywords: | immobilized pH gradient isoelectric focusing multichamber mixer two-dimensional technique |
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