Cellulose acetate zone electrophoresis

Figure 50-2. Technique of cellulose acetate zone electrophoresis. A A small amount of serum or other fluid is applied to a cellulose acetate strip. B Electrophoresis of sample in electrolyte buffer is performed.

See agarose, cellulose acetate, disc electrophoresis, isoelectric focusing, isotachophoresis, starch gel electrophoresis, zone electrophoresis... 

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. 

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work involved the use of filter paper as the supporting medium however, in recent years filter paper has been somewhat superseded by other materials, such as cellulose acetate, starch gel and polyacrylamide gel, which permit sharper separations. 

Agarose gel electrophoresis (typically 0.7-1 % agarose w/v) is increasingly used in clinical laboratories in place of cellulose acetate, for example in zone electrophoresis of serum proteins, isoenzyme analysis of lactate dehydrogenase and creatine kinase, and immunoelectrophoresis. 

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. 

Electrophoretic separation on agarose gels or cellulose acetate membranes is the procedure most commonly used to demonstrate LD isoenzymes." After the isoenzymes have been separated by electrophoresis, a reaction mixture is layered over the separation medium. The mixture (typically D, L-lactate> 500mmol/L, and NAD, 13mmol/L, often dissolved in a suitable pH 8.0 buffer) is applied as a liquid or in a gel. The NADH generated over the LD zones is detected either by its fluorescence, when excited by long-wave ultraviolet light (365 nm), or by its reduction of a tetrazolium salt to form a colored formazan. 

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. 

K29. Kohn, J., A cellulose acetate supporting medium for zone electrophoresis. Clin. Chim. Acta 2, 297-303 (1957). 

Electrophoretic properties of typical cellulase preparations, an extracellular cellulase from a culture on 0.5% cellulose and a cell-bound cellulase from that on 0.5% cellobiose, were compared in respect to their behavior in zone electrophoresis on cellulose acetate film. As shown in Figure 2, the former was separated into two components, A (fast moving to the cathode) and B (almost no moving). With the latter, a single component was detected under the same conditions. This fast moving component was in approximate agreement with component A in regard to its mobility, but as will be mentioned later, there was considerable difference in substrate specificity and other properties. Therefore, it seems to be a different component, and is referred to as component C. 

Purification and Physical and Chemical Properties. Extracellular cellulase components A and B and cell-bound cellulase component C were purified through the steps summarized in Figure 7 from the cultures of Ps. fluorescens on 0.5% Avicel and on 0.5% cellobiose, respectively. The purified cellulase components (Cellulases A, B, and C) thus obtained showed a single peak in zone electrophoresis on cellulose acetate film and starch bed. 

The movement of soil colloidal particles was the first description of electrophoresis as early as 1809. However, Arne Tiselius ( 1937) was the first to construct a successful instrument useful for the separation of serum protein by electrophoresis using the boundary separation principle. Because of the clinical significance of this type of separation, many improvements and refinements followed, such as utilizing paper, cellulose acetate, gel, and more recently capillaries in order to speed up and better separate (into distinct zones) the different proteins. The electric current can be utilized in the clinical applications to accomplish not just separation but other tasks ... 

The proteins are iisted in order of their eiectrophoretic mobilities at pH 8.6. The symbols a1, o2, 2, and y refer to zones of a given position in standard cellulose acetate electrophoresis and are correlated with their respective mobilities. Absolute values of intrinsic electrophoretic mobilities are not given here. M, male F, female. 

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... 

Wang, C.W. Chen, I.C. Cellulose acetate porous polymer joint for capillary zone electrophoresis. Anal. Chem. 1992 64, 2461-2464. 

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