Purification, electrophoretic

Classical gel electrophoresis has been used extensively for protein and nucleic acid purification and characterization [9, 10], but has not been used routinely for small molecule separations, other than for polypeptides. A comparison between TLC and electrophoresis reveals that while detection is usually accomplished off-line in both electrophoretic and TLC methods, the analyte remains localized in the TLC bed and the mobile phase is immediately removed subsequent to chromatographic development. In contrast, in gel electrophoresis, the gel matrix serves primarily as an anti-... 

The same principle is used for the preparative separation of mixtures of biological materials, the extraction of different individual components from these mixtures, and their purification. In this case one uses an electrophoretic method with continued introduction of individual portions of the mixture and withdrawal of portions of pure fractions. There have been reports that such processes were accomplished in spacecraft where, since gravitational forces are absent, the liquid solutions can be used without the danger of natural convection. 

Other purification methods include a liquid phase chromatography, electrophoretic separation by mass spectroscopy, separation using magnetic properties, and so on. These separation methods are limited only for the metal nanoparticles having a special property useful for these purification methods. 

Tejero-Diez, P., Rodriguez-Sanchez, P., Diez-Guerra, F.J. (1999). Microscale purification of proteins exhibiting anomalous electrophoretic migration application to the analysis of GAP-43 phosphorylation. Anal. Biochem. 274, 278-282. 

Coon, M.J., Ballou, D.P., Haugen, D.A., Krezoski, S.O., Nordblom, G.D. and White, R.E. Purification of membrane-bound oxygenases isolation of two electrophoretically homogeneous forms of liver microsomal cytochrome P-1+50. 

Isozymes are also a common presence in enzyme preparations and they can often be detected via polyacrylamide gel electrophoresis. The detected presence of isozymes may result in the need for further purification steps and the kinetic characterization of each isozyme. It may be necessary to use nondenaturing electrophoretic procedures to separate the different isozymes. See Isozymes Enzyme Concentration... 

In liquid chromatography, affinity purification protocols (4-8) have been known for a long time. Naturally, electrophoresis can be used just as well to observe molecular or noncovalent interactions of DNA oligomers, provided the complex has distinct electrophoretic properties different from those of the free molecules. Therefore, affinity capillary electrophoresis (ACE) can be a powerful tool for studying DNA-drug or DNA-biopolymer interactions. Several reviews discussing these aspects of ACE have been published in recent years (9-19). The crucial aspects of DNA in this field are covered comprehensively in a recent overview article (20). 

For rapid confirmation of the molecular mass of a recovered peptide following RPC purification, either ESI-MS or MALDI-MS procedures are now indispensable. Studies on the interaction of peptides with solvent molecules, ions, and free or immobilized ligands in chromatographic or electrophoretic environments have, however, been largely confined to methods that examine global properties of different peptides in the bulk mobile phase. In... 

Electrophoretic Methods. Several electrophoretic procedures have been developed to fractionate or purify the various caseins (McKenzie 1971C Thompson 1971 Whitney 1977). Wake and Baldwin (1961) fractionated whole casein by zone electrophoresis on cellulose powder in 7 M urea and 0.02 ionic strength sodium phosphate buffer at pH 7 and 5°C. Payens and co-workers employed several somewhat different electrophoretic conditions for the fractionation and purification of the caseins on cellulose columns (Payens 1961 Schmidt and Payens 1963 Schmidt 1967). Three fractions, as-, k-, and /3-caseins, were separated at pH 7.5 and 30°C with 4.6 M urea-barbiturate buffer. The purification of asi-casein and the separation of the genetic variants of K-casein were accomplished by altering the electrophoretic conditions. Manson (1965) fractionated acid casein on a starch gel column stabilized by a density gradient at 25 °C. 

As an analytical tool, electrophoresis is simple, relatively rapid, and has unparalleled resolving power. It is used chiefly for analysis and purification of very large molecules such as proteins and nucleic acids. Highly sensitive detection methods have been developed to monitor and analyze electrophoretic separations. 

The purification of glutamine cyclotransferase from papaya latex has been carried out by Messer and Ottesen (116). A batch procedure was used for the removal of impurities by passage of a papaya latex extract through a thin layer of carboxymethyl-Sephadex the active protein was separated by selective elution. Additional purification was achieved by chromatography on a column of carboxymethyl-Sephadex and by gel filtration on Sephadex G-100. The purified enzyme was homogeneous by the criteria of paper electrophoresis, ultracentrifugation, and gel filtration on Sephadex G-100 columns and chromatography on carboxy-methyl- or DEAE-Sephadex. The electrophoretic behavior of the enzyme indicates that it is a basic protein with an isoelectric point near... 

Although electrophoresis is the method of choice for assessing protein purity, it is not frequently used as a purification step, at least not yet. This is because of the small amounts that can usually be analyzed and because of the denaturing conditions that are often used in electrophoretic analysis. 

Particle electrophoresis studies have proved to be useful in the investigation of model systems (e.g. silver halide sols and polystyrene latex dispersions) and practical situations (e.g. clay suspensions, water purification, paper-making and detergency) where colloid stability is involved. In estimating the double-layer repulsive forces between particles, it is usually assumed that /rd is the operative potential and that tf/d and (calculated from electrophoretic mobilities) are identical. 

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