Fractionation of Complex Proteomes by Microscale Solution Isoelectrofocusing Using ZOOM− IEF Fractionators to Improve Protein Profiling
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All current methods for quantitatively comparing protein profiles, including twodimensional polyacrylamide gel electrophoresis (2-D PAGE) as well as non-2-D gel methods such as isotope-coded affinity tag methods (ICAT) and other liquid chromatography (LC)/ tandem mass spectrometry (MS/MS) methods, have limited resolution and modest dynamic ranges for detection (1, 2, 3, 4, 5). As a result, these proteomic methods can analyze only small portions of very complex proteomes, such as cell or tissue extracts from higher eukaryotes. One strategy to increase the proportion of complex proteomes that can be quantitatively compared by existing protein-profiling methods is to fractionate these samples into a modest number of well resolved pools prior to downstream multi-dimensional protein profiling using 2-D gels or LC/LC-MS/MS methods. Unfortunately, most protein separation methods have relatively low resolving power and often do not separate noncovalent complexes. As a result, low- to moderate-resolution fractionation methods typically result in extensive and variable cross-contamination of many proteins among multiple fractions, which makes quantitative comparisons impractical. In contrast, several recently developed, high-resolution, liquid-phase isoelectric focusing (IEF) methods can fractionate proteomes into a modest number of well-separated fractions under denaturing conditions where specific and nonspecific protein-protein interactions are minimized (6, 7, 8, 9, 10).
KeywordsBioRad Laboratory Carrier Ampholyte Separation Chamber Partition Membrane Complex Proteomes
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