Electrophoresis on cellulose acetate

The isoenzymes can be separated by electrophoresis on cellulose acetate, and Roberts, et al ( ) have described a method whereby the separated isoenzymes are eluted and then assayed kinetically. 

CK isoenzymes have been separated by electrophoresis on cellulose acetate, agar gel, agarose and polyacrylamide gel. 

Electrophoresis on cellulose acetate strips (Sepraphore III, Gelman Instrument, Ann Arbor, MI) was done in the conventional manner [12] in order to obtain a comparative electrophoretic mobility of non-adsorbed albumin. For this purpose, BSA-BSA (2.5 w/v) was deposited on the cellulose acetate paper twice in volumes of 10 ul each. Electrophoresis was attain performed in the Gelman Chamber with Pt electrodes at 20°C (see Table 3) After completion, the strips were stained with Ponceau S protein stain (Gelman Instruments) and washed with 5 acetic acid. The stained cellulose acid strips were subsequently cut into 3 mm wide pieces which were monitored for protein content y-count-ing. 

Electrophoresis has also been employed to separate neomycin from analytically-interfering substances such as proteins. Hence Brammer and Hemsonl82 have determined the neomycin content of blood serum. Neomycin was separated from the serum proteins by electrophoresis on cellulose acetate and assayed colorimetrically following elution from the support. 

Procedure 11.7 Location of proteins after electrophoresis on cellulose acetate... 

W5. Windisch, R. M., and Bracken, M. M., Cerebrospinal fluid proteins Concentration by membrane ultrafiltration and fractionation by electrophoresis on cellulose acetate. Clin. Cherru 16,416-419 (1970). 

Before discussing the various derivatives that have been used, it should be remembered that oligosaccharides are often obtained by chromatography on paper or on cellulose columns, and this may cause them to become contaminated by xylan this may be eliminated by extraction of the crude fractions with hot ethanol.474 For similar reasons, glucose that is really extraneous may be detected in fractions separated by electrophoresis on cellulose acetate.475 Gas-liquid chromatography was used to show the presence in pituitary glyco-... 

Electrophoresis on cellulose acetate strips has also been used for the rapid resolution of whey proteins (Bell and Stone 1979). Samples of a 10 1 concentrate of whey are applied to cellulose acetate strips which have been saturated with Tris-barbiturate buffer, pH 8.6, ionic strength 0.097, and the electrophoresis is performed at 225 V for 1 hr. This procedure separates not only the major whey proteins but also their genetic variants. 

Standard electrophoresis on cellulose acetate and citrate agar. 

Fig. 10. Hybridization of GAPDH s (a) rabbit (R) and lobster (L) muscle, pig (P) and lobster (L) muscle (b) rabbit (R) and yeast (Y), pig (P) and yeast (Y). The hybrid bands were separated by electrophoresis on cellulose acetate in 50 mM phosphate buffer, pH 7.0, and revealed in (a) by protein staining and in (b) by activity staining (cf 103).

Borate buffer, introduced in our laboratory for paper electrophoresis of gastric juice (G19) has since been used for this purpose by other authors (BI2, C5a, Dl, FI, G33, G34, HI, K2, Via, Wll) and also for electrophoresis on cellulose acetate strips (P3, P4). Instead of vertical paper electrophoresis units, some of these investigators used borate buffers with horizontal units (B13, FI, K2, Via). The resolution obtained was certainly less satisfactory than that yielded by the vertical unit (Via) and cathodic peaks were not resolved (B13, C5a, K2). 

Piper et al. (P3, P4) applied the paper electrophoretic method of our laboratory to gastric juice electrophoresis on cellulose acetate strips. They collected gastric juice after augmented histamine stimulation, but gastric acidity prior to collection was neutralized in situ by intragastric instillation of sodium bicarbonate in 5-20% solution. 

Electrophoresis is a possible procedure for fractionating sequence isomers since the resistance to motion will be affected by secondary structure and charge distribution as well as by any effect due to differences in total charge. This can be seen on cellulose acetate at pH 3.5 by the separation of the tetranucleotides Gp(Gp, Ap)Up for example (Sanger et al. 1965). Electrophoresis on DEAE-paper in 7% formic acid is particularly powerful, and admirably complements electrophoresis on cellulose acetate at pH 3.5. For example, ApGpGpUp is separated from Gp(Gp, Ap)Up (Brownlee 1972). 

Figure 11.5 Migration patterns of a mixture of five Hb species (a) by IEF over the pH 6-9 range, and (b) by electrophoresis on cellulose acetate strips at alkaline pH.8 [Reprinted, with permission, from P. Basset, F. Braconnier and J. Rosa, J. Chromatogr. 227, 1982, 267-304. An Update on Electrophoretic and Chromatographic Methods in the Diagnosis of Hemoglobinopathies . 1982 Elsevier Publishing Company.]...

Fig. 14. Malignant paraproteinemia. Electrophoresis on cellulose acetate of serum (above) and concentrated urine (below) reveals (i) Bence Jones proteinuria. In this case monoclonal bands are seen of each type. For L the relevant concentration to albumin indicates which are IgGL, L dimer, and L monomer (ii) loss of normal y-globulin (iii) a high serum level of paraprotein. Immunoelectrophoresis of urine reveals paraprotein bows of k, and X, yaXj and X2.

Fig. 15. Benign paraproteinemia. Electrophoresis on cellulose acetate of serum and concentrated urine reveals (i) no Bence Jones proteinuria (ii) no loss of normal y-globulin (iii) a low level of serum paraprotein. In this case the paraprotein is of post-y-mobility, is type GL, and is typically (but not exclusively) found in lichen myxedematosus. Reproduced by courtesy of the Proceedings of the Royal Society of Medicine (H30).

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. 

F23. Friedman, H. 8., A standardized procedure for serum protein electrophoresis on cellulose acetate membrane strips. Clin. Chim. Acta 6, 775-781 (1961). 

Another modification in which form thin sheet electrophoresis is employed is known as ionophoresis. In this method, devised by Sanger and his co-workers, high voltage electrophoresis is carried out on ion-exchange paper. It is a rapid method of great resolution and sensitivity and is used in Biochemistry for the separation of constituents of a highly complex mixture e.g. a mixture of oligoribonucleotides produced by partial enzymic hydrolysis of RNA. For separation of the constituents of this hydrolysate mixture, a two-dimensional technique is used in which the mixture is subjected to electrophoresis on cellulose acetate and then the partially separated mixture is transferred to a DEAE-cellulose... 

Hemoglobin variants have been typed by conventional electrophoresis on cellulose acetate or agarose and by lEF (1, 52, 66-70). Budowle and Eberhardt (5 ) recently developed an ULPAGIF method for typing the A, F, S, C and a number of rare variants, which is presently used for the analysis of bloodstained evidence submitted to the FBI Laboratory. The method employs pH 6.7-7.7 Pharmalytes in an ultrathin-layer gel with an inter-electrode wick distance of only 5.0 cm. The result is a rapid screening method for Hb that takes only 25-30 minutes-comparable to cellulose acetate. Further, the distances between the A-F, F-S and S-C were 4 oim, 7 mm and 12 mm, respectively, compared with 3 mm, 5.5 nim, and... 

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. 

A search for lysosomal hydrolases and related enzymes has been made in haemolysates from human and rabbit red cells. Apart from acid phosphatases, significant activities were found only for a-D-mannosidase, neutral o-D-glucosidase, and -D-2-acetamido-2-deoxyhexosidase. a-D-Mannosidase activity per cell in human red blood cells was 200-times lower than in white cells. The optimal pH was 5.5-6.0. Electrophoresis on cellulose acetate showed three bands. Haemolysates from four patients with mannosidosis were not deficient in a-D-mannosidase. Curves of pH activity and electrophoretic patterns were similar to those of controls. From its biochemical and genetic properties, it is concluded that red cell a-D-mannosidase differs from the lysosomal acid a-D-mannosidase. 

Electrophoresis on cellulose acetate once widely used from separating serum proteins has been replaced by commercial high-resolution gel electrophoresis. 

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