Proteins of interest may be cut from stained gels and identified by MS. The gel patterns may be compared by eye or digitized images compared using commercial 2D software such as Progenesis SameSpots from TotalLab. The resulting starburst pattern of protein spots may be visualized on final 2D gels by autoradiography, protein stains such as silver, Coomassie and Sypro Ruby, or by western blotting (WB). Proteins are separated by charge in the first dimension using isoelectric focusing (IEF) then by size using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE). Two-dimensional electrophoresis (2D PAGE), in place since 1975, is a biochemical method for separating complex mixtures of proteins into individual species. ![]() Examples of biomedical applications are provided in which 2D proteins of interest found by comparing stained or western blotted 2D gel patterns were identified by mass spectrometry (MS). Quantification of 60 proteins in rat liver cytosol over a wide range of pI and MW gave linear plots of spot density versus total protein for loads of 200, 400 and 600 μg protein dissolved in SDS buffer and run in triplicate on 2D gels (Average R 2 = 0.987). The 2D gel pattern of rat liver microsomes shows more detail and sharper spot outlines when dissolved in SDS buffer with heating than in urea buffer and is better yet when dissolved in a mixture of both buffers. ![]() Evidence is provided that 2D SDS PAGE is reproducible, robust and compatible with SDS in both dimensions including isoelectric focusing in tube gels, the first dimension. Two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (2D SDS PAGE) is a method that separates proteins according to their isoelectric points in the first dimension and molecular masses in the second dimension.
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