Isoelectric focusing development

Another recent technique, preparative electrophoresis, is recycling isoelectric focusing developed by Bier and colleagues (University of Arizona). See Fig. 2. 

Palm A. (2003) Capillary isoelectric focusing developments in protein analysis. In G Marko-Varga and P Oroszlan (eds). Emerging Technologies in Protein and Genomic Material Analysis. Journal of Chromatography Library, Vol. 68, pp. 118-122 209-210. Elsevier Science, New brk. 

First Dimension Optimization After the second-dimension separation has been developed, the first-dimension flow rate is determined. This includes selecting a first-dimension column diameter to work at the flow rate selected. We illustrate the selection process with an application that addresses a column method for proteins that functions as a replacement for planar 2D gel electrophoresis (2DGE) within a narrow molecular weight and p/range. In the planar experiment, isoelectric focusing is performed in the first dimension and sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS/PAGE) in the second dimension. 

The following procedure relates to electrophoretic protocols where the first dimension is developed by isoelectric focusing (in tube gels) and the second dimension is a size exclusion separation by SDS polyacrylamide electrophoresis in a slab gel. 

Purity and homogeneity of the purified protein is assessed by macromolecular exclusion chromatography, SDS-polyacrylamide gel electrophoresis, isoelectric focusing, and matrix-assisted laser desorption/ionization-time of flight mass spectrometry. The later technique, developed by Karas and Hillenkamp, ionizes and separates proteins on the basis of their mass-to-charge ratio (Karas and ffillenkamp, 1988). 

Figure l shows the gold stain of a section of a 2D gel of beef heart mitochondria proteins separated by isoelectric focusing and then by SDS-PAGE, and transferred to nitrocellulose. The filter was then probed with antibody specific to the 49-kDa iron sulfur protein of NADH CoQ reductase. The blot was photographed with (Fig. 2) and without (Fig. 3) a red filter following development with 4-chloro-1 -naphthol. The position of the 49-kDa protein can then be determined by back reference to Fig. 1 (see arrow). 

Among the electrophoretic methods of chiral resolution, various forms of capillary electrophoresis such as capillary zone electrophoresis (CZE), capillary isotachophoresis (CIF), capillary gel electrophoresis (CGE), capillary isoelectric focusing (CIEF), affinity capillary electrophoresis (ACE), and separation on microchips have been used. However, in contrast to others, the CZE model has been used frequently for this purpose [44]. On the other hand, drawbacks associated with the electrophoretic technique due to lack of development of modem chiral phases have limited the application of these methods. Moreover, the electrophoretic techniques cannot be used at the preparative scale, which represents an urgent need of chiral separation science. 

The development of electrophoretic techniques afforded possibilities for fractionations based on charge density differences. Duxbury (1989) has reviewed applications of different electrophoretic separation methods, including zone electrophoresis, moving boundary electrophoresis, isotachophoresis, and isoelectric focusing (IEF). Preparative column electrophoresis (Clapp, 1957) and continuous flow paper electrophoresis (Hayes, 1960 summarized by Hayes et al., 1985) methods have been used to separate components isolated from sapric histosol soils. These techniques allowed separation of polysaccharides from the colored components the electrophoretograms of the colored components were diffuse, showing a continuum of components of different charge densities. 

Chapter 5 includes sufficient details on IEF to provide a better understanding of this technique. It also includes a number of applications of various proteins. Isoelectric focusing is applicable only to the fractionation of amphoteric species, such as proteins and peptides, that can act both as acids and bases. Nonamphoteric species, nucleic acids in particular, cannot be resolved by IEF. Both analytical and preparative modes of IEF, included in this chapter, have been developed as valuable tools for studying proteins. 

CE has many separation modes that are beneficial to protein impurity analysis. Within the many thousands of potential protein impurities in a recombinant product there will be several that have only minor physicochemical differences from the drug product. The application of different CE modes can potentially resolve these impurities. CE methods can be divided into four principle modes that are applicable to recombinant protein impurity analysis capillary zone electrophoresis, capillary isoelectric focusing, capillary gel electrophoresis, and micellar electrokinetic capillary chromatography. Each mode will be discussed briefly. Since the technology is so young and still very exploratory, CE methods are developed empirically for specific separations. It is difficult to provide standard protocols for CE impurity analysis. Instead, protocols that can be used as a starting point for impurity analysis will be provided as well as the citation of examples of impurity analyses from the literature to provide additional sources of protocols for interested readers. 

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