By Anton Posch
This publication offers huge insurance of the foundations and up to date advancements of pattern training and fractionation instruments in Expression Proteomics in most cases and two-dimensional electrophoresis (2-DE) particularly. With its specific means to unravel millions of proteins in one run, 2-DE continues to be a primary study instrument for almost all protein-related medical projects.
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Extra info for 2D PAGE: Sample Preparation and Fractionation
Because the cell lysate usually remains in the sample solubilization buffer for up to 1 hour to fully denature, disaggregate, and solubilize the proteins, it is important to inactivate the aforementioned enzymes (in particular proteases) as rapidly and effectively as possible to prevent protein degradation or modification that otherwise may result in artifactual spots and loss of high molecular mass proteins. Because enzymes are less active at lower temperatures, sample preparation at as low a temperature as possible is recommended, preferably at –196 °C (liquid nitrogen).
Sample preparation and solubilization are crucial factors for the overall performance of the 2D PAGE technique. Protein complexes and aggregates should be completely disrupted in order to avoid appearance of new spots because of a partial protein solubilization. Persistent cloudiness of the lysate may indicate incomplete lysis. Most methods available carry out lysozyme digestion on ice, however, if your protein(s) is fairly thermostable, it is recommended to perform the digestion at 25–30 ºC as this is the optimal temperature for lysozyme.
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2D PAGE: Sample Preparation and Fractionation by Anton Posch