Isoelectric focusing and two-dimensional electrophoresis of tubulin using immobilized pH gradients under denaturing conditions |
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Authors: | D J Field J C Lee |
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Institution: | 1. Department of Earth and Atmospheric Sciences, University of Alberta, T6G 2E3 Edmonton, AB, Canada;2. Institut fur Geochemie und Petrologie, ETH Zurich, 8092 Zurich, Switzerland;3. Department of Geosciences, Mohammed Premier University, Oujda, Morocco |
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Abstract: | Modifications of the LKB Immobiline isoelectric focusing (IEF) technique are described for use under conditions that solubilize and denature most proteins (8 M urea and 2% Nonidet-P40). This procedure permits pH gradients that are four- to fivefold shallower than previously available with conventional ampholine-IEF procedures. It can also be used as a first dimension in two-dimensional gel electrophoresis. The advantage of the stable ultranarrow pH gradient is demonstrated by directly comparing the resolution of vertebrate brain tubulins using (i) denaturing conventional ampholine-IEF and (ii) denaturing Immobiline-IEF. Analysis of tubulin on the Immobiline-IEF gel increases the separation distance between the individual tubulins and distinguishes differences among tubulin samples that could not be resolved by conventional ampholine isoelectric focusing. |
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