Zone Electrophoresis in Starch Gels and

Smithies, O., Zone electrophoresis in starch gels and its application to studies of serum proteins. Advances in Protein Chem. 14, 65 (1959). 

Zone Electrophoresis in Starch Gels and Its Application to Studies of Serum Proteins O. Smithies... 

The pectinesterase produced by Sclerotinia libertiana78 was purified on columns of Duolite A-2, Amberlite CG-50, and CM-cellulose. The final product was purified 266-fold, its sedimentation coefficient was calculated to be 4.41 S, and zone electrophoresis in starch gel showed a slight contamination of this product. 

H23. Hunter, R. L., and Markert, C. L., Histochemical demonstration of enzymes separated by zone electrophoresis in starch gels. Science 126, 1294-1295 (1957). 

Zone electrophoresis is used mainly as an analytical technique and, to a lesser extent, for small-scale preparative separations. The main applications are in the biochemical and clinical fields, particularly in the study of protein mixtures. Like chromatography, zone electrophoresis is mainly a practical subject, and the most important advances have involved improvements in experimental technique and the introduction and development of a range of suitable supporting media. Much of the earlier work involved the use of filter paper as the supporting medium however, in recent years filter paper has been somewhat superseded by other materials, such as cellulose acetate, starch gel and polyacrylamide gel, which permit sharper separations. 

Isoenzymes have usually been separated by zone electrophoresis on various carriers and identified by subsequent specific staining. Starch, agar, agarose, and polyacrylamide gels, cellulose acetate foils, and dex-trans such as Sephadex are the most commonly used carrier media. Markert and Mpller (1959) were the first to apply the technique of starch gel electrophoresis in separating dehydrogenases. Apart from the fact that such electrophoreses are easily carried out, they require only... 

On an analytical scale, one can separate the L and H chains, after cleavage of interchain disulfide bonds, by zone electrophoresis in the presence of a dissociating agent, e.g., concentrated urea or detergent (2,8). (For example, in urea-starch gel at low pH, L chains move more rapidly than H chains toward the cathode.) Conventional or disc electrophoresis in polyacrylamide gel has also been used frequently for analytical purposes (9,10). 

In another investigation (Neelin and Butler, 1961) specificity and a high degree of heterogeneity of histones were also discovered in the organs of the chicken. Histones of the spleen and liver, when tested by zonal electrophoresis on starch gel, formed 18 zones, the arrangement of which varied. Histones of the erythrocytes, however, possessed a much smaller assortment of components. 

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. 

Zone electrophoresis is normally carried out horizontally in a suitable medium such as paper, polyacrylamide gel, starch gel or cellulose acetate. The sample components can be completely separated and quantitatively and qualitatively identified in much lower quantities than by the moving-boundary method. The procedure consists of saturating the support material with a buffer solution. The ends of the strip of support are immersed in separate reservoirs of buffer solution to maintain the saturation. The sample is then applied as a narrow band near one end of the support strip. A voltage potential is created down the length of the strip causing the sample components to ionize and then migrate at a rate dependent on their charge, molecular size and interactions with the support medium. When the process is complete, the strip is removed and developed for examination of the separated components. Densitometry is normally used for quantitation of the bands after suitable color development. 

Dextran gels and other gel substances mentioned can be used as support in electrophoresis. It should be of great value to find out if similar separations can be obtained with gel filtration supplemented with pure zone electrophoresis as with starch-gel electrophoresis according to Smithies (1955). 

Sur et al. (S31) subjected a concentrated aqueous extract of human prostate gland to starch gel electrophoresis in citrate buffer at pH 6.2, and obtained at least thirteen active zones. These were recovered from... 

The support medium provides the matrix in which protein separation takes place. Various types of support media are used in electrophoresis and range from pure buffer solutions in a capfilary to insoluble gels (e.g., sheets, slabs, or columns of starch, agarose, or polyacrylamide), or membranes of cellulose acetate. Gels are cast in a solution of the same buffer to be used in the procedure and may be used in a horizontal or vertical direction. In either case, maximum resolution is achieved if the sample is applied in a very fine starting zone. Separation is based on differences in charge-to-mass ratio of the proteins and, depending on the pore size of the medium, possibly molecular size. 

Fig. 21. Alkaline phosphatase zones after starch-gel electrophoresis of human intestinal extracts. The single pattern on the left labeled d is that of fresh butanol extract of human intestine [from Fig. 1 in Moss report (M34)]. The pattern labeled E is the same intestinal extract stored for 4 months at —20°C (before chromatography). A, B, and C are different fractions obtained by DEAE-cellulose chromatography of E (M34).

Bourne (S3).] Moss and King (M36) separated different zones of human alkaline phosphatase by starch-gel electrophoresis and determined their Michaelis constants. They also found a high degree of overlap of the various bands on starch-gel electrophoresis of purified human tissue alkaline phosphatases. In another study, purified intestinal alkaline phosphatase (M34, M35) was found to travel to several positions including the typical slow zone. More recently Moss et al. (M38) reported the neuraminidase sensitivity of human liver alkaline phosphatase. Butter-worth and Moss (B45) showed that purified human renal alkaline phosphatase is also neuraminidase-sensitive, as the electrophoretic mobility of the enzyme in the starch gel was considerably reduced after neuraminidase treatment. 

In our opinion, it is unwise to base a classification of isoenzymes solely on starch-gel electrophoresis data. For example, the work of Boyer, Chiandussi, and many others (B39, C7, M33-M36) shows a great complexity in the location of bands derived from individual organs. True, one can identify the heat-stable or -unstable zones and even those... 

Electrophoresis on cellulose acetate paper has also been employed for the characterization of human alkaline phosphatase isoenzymes by Korner (K20, K21) and Posen et al. (P19). A modification of the Gomori technique (G11-G13) for the histochemical localization of alkaline phosphatase was made by Allen and Hyncik (AlO) to visualize the enzyme zones in the starch and agar gels. 

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