Isoelectric focusing, high-resolution protein analysis

Table 3.2 summarizes the most popular proteome profiling techniques. Recent developments introduced capillary electrophoresis [92] not only for DNA analysis and sequencing, but also for high-resolution protein separation, based on their size (capillary electrophoresis-sodium dodecyl sulfate [CE-SDS]) and capillary isoelectric focusing (cIEF) point... 

Isoelectric focusing is a mature separation technique that has a place in any laboratory doing work with proteins. The analysis of a protein is not complete without a determination of its isoelectric point and all protein databases have at least estimates of the pis of the represented proteins. Proteins thought to be pure by other methods are often found to be mixtures of several proteins when analyzed by IEF. Isomeric forms of the same protein that are revealed by IEF are valuable indicators of mutations or differences in posttranslational modifications. IEF plays a crucial role in 2-D PAGE and preparative IEF allows for high-purity fractionations of unparalleled resolution. A simple keyword search of literature databases shows that about 500 journal articles are written per year referring to IEF. This amply attests to the value of IEF as a tool for protein analysis and purification. 

Capillary isoelectric focusing (CIEF) is a high-resolution technique for protein and peptide separation performed at academic sites and in the biotechnology and pharmaceutical industries for the analysis and characterization of, for example, recombinant antibodies and other recombinant proteins, isoforms of glycoproteins, point mutations in hemoglobin, and peptide mapping. Also, hyphenation to mass spectrometry and chip-based CIEF (microfabrication) have shown promise. CIEF kits and specific recipes/application notes are available from vendors of capillary electrophoresis (CE) equipment, as are a vast amount of publications and handbooks of CE published over recent years. 

Wei, J., L. Yang, A. K. Harrata, and C. S. Lee, High resolution analysis of protein phosphorylation using capillary isoelectric focusing—electrospray ionization—mass spectrometry. Electrophoresis, 19, 2356-2360, 1998. 

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