Isoelectric focusing applications

In considering the applicability of preparative classical electrophoretic methods to chiral separations, it should be noted that practitioners in the art of classical electrophoresis have been particularly inventive in designing novel separation strategies. For instance, pH, ionic strength and density gradients have all been used. Isoelectric focusing and isotachophoresis are well-established separation modes in classical electrophoresis and are also being implemented in CE separations [7, 8]. These trends are also reflected in the preparative electrophoretic approaches discussed here. 

Righetti, P. G., Isoelectric Focusing Theory, Methodology and Applications, Elsevier, New York, 1983. 

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. 

Two-dimensional electrophoresis is normally run so that proteins are separated from each other on the basis of a different molecular property in each dimension. The most commonly utilized method entails separation of proteins by isoelectric focusing (see below) in the first dimension, with separation in the second dimension being undertaken in the presence of SDS, thus promoting band separation on the basis of protein size. Modified electrophoresis equipment that renders two-dimensional electrophoretic separation routine is freely available. Application of biopharmaceuti-cal finished products to such systems allows rigorous analysis of purity. 

Isoelectric focusing also finds application in analysing the stability of biopharmaceuticals over the course of their shelf life. Repeat analysis of samples over time will detect deamidation or other degradative processes that alter protein charge characteristics. 

FIGURE 12 Application of capillary isoelectric focusing (clEF) for the determination of apparent p/ values of rMAb samples. Capillary Bio-Rad Bio-CAP XL capillary (50 pm x 24 cm) ampholyte 80% clEF Bio-Lyte Ampholyte 3-10 (2% solution with 0.5% TEMED, 0.2% HPMC) anolyte 20 mM phosphoric acid catholyte 40 mM sodium hydroxide focusing l5kV (625V/cm) for 5 min mobilization 20 kV (833V/cm) for 25 min with zwitterions (cathodic mobilizer from Bio-Rad) capillary temperature 25°C. (Reprinted from reference 40, with permission.)... 

Staugaitis, S. M., Shapshak, R, Tourtellotte, W. W., etal. Isoelectric focusing of unconcentrated cerebrospinal fluid Applications to ultrasensitive analysis of oligoclonal immunoglobulin G. Electrophoresis 6, 287-291 (1985). 

Use a suitable apparatus connected with a recirculating temperature-controlled water bath set at 10°C and gels for isoelectric focusing with a pH gradient of 3.5-9.5. Operate the apparatus in accordance with the manufacturer s instructions. Use as the anode solution phosphoric acid R (98 g/1 H3PO4) and as the cathode solution 1 M sodium hydroxide. Samples are applied to the gel by filter papers. Place sample application filters on the gel close to the cathode. 

Apply 15 /il of the test solution and 15 /il of the reference solution. Start the isoelectric focusing at 1500 V and 50 mA. Turn off the power after 30 min, remove the application filters and reconnect the power supply for 1 h. Keep the power constant during the focusing process. After focusing, immerse the gel in a suitable volume of a solution containing 115 g/1 of... 

Allen RC, Saravis CA, Maurer HR (1984) Gel electrophoresis and isoelectric focusing of proteins. Selected techniques. W. de Gruyter, Berlin Chrambach A, Dunn MJ, Radola BJ (eds.) (1987 foil.) Advances in electrophoresis, vol. 1 foil., VCH, Weinheim Hames BD, RickwoodD (eds.) (1990) Gel electrophoresis of proteins a practical approach, 2nd ed., Oxford University Press, New York Deyl Z, Chrambach A, Everaerts FM, PrusikZ (eds.) (1983) Electrophoresis. A survey of techniques and applications. J Chromatogr Library, vol. 18B, Elsevier, Amsterdam... 

Zone electrophoresis is influenced by adsorption and capillarity, as well as by electroosmosis. Therefore evaluation of mobility (and f) from this type of measurement is considerably more complex than from either microelectrophoresis or moving-boundary electrophoresis. Nevertheless, zone electrophoresis is an important technique that is widely used in biochemistry and clinical chemistry. One particularly important area of application is the field of immunoelectrophoresis, which is described briefly in Section 12.11. Additional information on zone electrophoresis may be obtained from Probstein (1994) and Hunter (1981) and the references given there. Variants of zone electrophoresis also exist see, for example, Gordon et al. (1988) for information on a variant known as capillary zone electrophoresis and Righetti (1983) for information on what is known as isoelectric focusing. 

Catsimpoolas, N., and Drysdale, J., eds. (1977) Biological and Biomedical Applications of Isoelectric Focusing, Plenum, New York... 

Isoelectric focusing takes along (from ca 3 to 30 h) time to complete because sample compounds move more and more slowly as they approach the pH in the gel that corresponds to their isoelectric points. Because the gradient ampholytes and the samples stop where they have no mobility, the resistivity of the system increases dramatically toward the end of the experiment, and the current decreases dramatically. For this reason, isoelectric focusing is usually mn with constant voltage. Constant current application can lead to overheating of the system. 

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. 

The following table provides a list of proteins that may be used as internal standards, along with their isoelectric points, pi, in quantitative applications of polyacrylamide gel electrophoresis. These proteins may be used in isoelectric focusing or in SDS-PAGE. The isoelectric points are reported at 25°C.1... 

B. Michalke, P. Schramel, Application of capillary zone electrophoresisDinductively coupled plasma mass spectrometry and capillary isoelectric focusing-inductively coupled plasma mass spectrometry for selenium speciation, J. Chromatogr. A, 807 (1998), 71D80. 

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