Isoelectric focusing preparative electrophoresis

There are three distinct modes of electrophoresis zone electrophoresis, isoelectric focusing, and isotachophoresis. These three methods may be used alone or in combination to separate molecules on both an analytical (p.L of a mixture separated) and preparative (mL of a mixture separated) scale. Separations in these three modes are based on different physical properties of the molecules in the mixture, making at least three different analyses possible on the same mixture. 

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. 

Preparative electrophoresis on Sephadex G-25 (Ref. 168) or double isoelectric focusing,208 preceded by chromatography on Sephadex G-75, CM-cellulose, and calcium phosphate, was used for the isolation of endo-D-galacturonanase from the filtrate of a Verticillium albo-atrum culture. The homogeneity was confirmed in both cases by electrophoresis on poly(acrylamide) gel. The molecular weight of the enzyme was close to the values found for Aspergillus endo-D-galacturonanases. 

Electrophoretic Methods Systems for polyacrylamide gel electrophoresis, 104, 237 preparative isoelectric focusing,104, 256 gel... 

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

Fully equipped protein purification laboratories should also have preparative electrophoresis and isoelectric focusing apparatuses for rare occasions when other techniques fail to give sufficient separation. 

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. 

Electrophoresis plays a key role as an analytical or preparative technique in the characterization of natural organic matter because it gives information about the behavior of these molecular mixtures in controlled solution conditions, depending on both the size and the charge distribution frequency of the analytes in the complex mixture. Historically, the first electrophoretic separations were conducted with environmental colloids and over the years all the techniques based on zone, gel electrophoresis, or isoelectric focusing were used in their different setups to analyze natural organic matter and environmental particles to a minor extent. The goal of... 

Masuoka, J., Glee, P.M., and Hazen, K.C. (1998). Preparative isoelectric focusing and preparative electrophoresis of hydrohobic Candida albicans cell wall proteins with in-line transfer to polyvinylidene difluoride membranes for sequencing. Electrophoresis 19, 675-678. 

Usually, immunoglobulins are N-glycosylated at the Fc part in some cases, terminal neuraminic acid may be missing. This leads to slightly different isoelectric points of the various members in the family called isoforms. Therefore, an IgG preparation which exhibits a single band in sodium dodecyl sulfate (SDS) electrophoresis exhibits a characteristic band pattern in isoelectric focusing (Fig. 4). 

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