Prefractionation of Complex Protein Mixture for 2-D PAGE Using Reversed-Phase Liquid Chromatography
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In the last years, great progress has been achieved by development of multidimensional liquid chromatographic separation methods for separation of protein complexes after enzymatic digestion and subsequent identification of the proteins with mass spectrometric techniques (1, 2, 3, 4). However, high-resolution two-dimensional gel electrophoresis (2-DE) is until now the only technique that allows separation of thousands of proteins in a gel (5, 6, 7, 8). It has been estimated that the proteome of a given cell contains at least 10,000-30,000 different proteins, but only 2000–10,000 proteins can be visualized on a silver-stained 2-DE gel, depending on the 2-DE method applied (8), and only a proportion of them are present at levels sufficient for mass-spectrometric identification. The introduction of two-dimensional differential gel electrophoresis (2-D DIGE) samples on a single 2-DE gel. Moreover, the dynamic range of the covalent fluorescent protein staining is higher than silver staining. This increases the reproducibility and saves material and time of the experiment.
KeywordsProtein Spot Mass Spectrometric Technique Carrier Ampholyte Human Breast Epithelial Cell Line Number Gene Product
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