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| Year : 1984 | Volume
: 30
| Issue : 3 | Page : 171-3 |
Split gel technique of polyacrylamide gel disc electrophoresis for comparing two biological fluids.
Yemul VL, Saoji AM, Jad CY, Kelkar SS
How to cite this article:
Yemul V L, Saoji A M, Jad C Y, Kelkar S S. Split gel technique of polyacrylamide gel disc electrophoresis for comparing two biological fluids. J Postgrad Med 1984;30:171 |
How to cite this URL:
Yemul V L, Saoji A M, Jad C Y, Kelkar S S. Split gel technique of polyacrylamide gel disc electrophoresis for comparing two biological fluids. J Postgrad Med [serial online] 1984 [cited 2023 Oct 1];30:171. Available from: https://www.jpgmonline.com/text.asp?1984/30/3/171/5456 |
Polyacrylamide gel disc electrophoresis (PAGDE) is one of the most elegent methods of separating mixtures of macromolecules in biological fluids. This is because of the molecular sieving effect in addition to the usual electrophoretic resolution. A sample of human serum may show as many as thirty components. This resolving capacity creates problems with identification of individual bands. Comparisons of duplicate separations in two tubes are not easy because of slight differences in electrophoretic mobilities and variables of each gel. A careful survey of the literature shows two isolated reports of a novel method that makes possible a study of two samples simultaneously in PAGDE. Clarke[1] achieved this by inserting a partition at the spacer gel level so that two reservoirs were created. Spencer and Guest[4],[5] used the method to compare inner membrane of Escherichia coli. These authors have not described the technique in adequate details which would facilitate its use more often. Clarke[1] used a thick waxed paper partition which we found rather unsatisfactory to obtain a desirable split gel effect. Spencer and Guest[4],[5] used a polysterene partition and made only a passing reference to the technique. This may be the reason why there is a paucity of literature on the subject.
We describe the technique of split gel in greater details and our experiments in standardizing the method, which will enable a novice embarking on PAGDE to reproduce the same without problem.
Lot of attention has been paid for the optimization of the technique by using various combinations of biological fluids such as human serum, CSF and the bacterial sonicates. In addition to the conventional staining procedures, special staining procedures were also employed for the demonstration of different components. This has made possible to get correct insight and realization of the real potential of the technique and enable many workers commonly using PAGDE to make comparisons between standards and unknown mixtures.
The method of PAGDE used was essentially that of Davis.[2] The only modification was in the glass tube used to cast gel [Fig. 1]. This was the insertion of a glass partition, 4 cm x 6 mm cut from a 1.5 mm thick glass slide, the edge's carefully ground and sealed with the epoxy resin adhesive "Araldite". In preparing the gel the separator was cast first upto the bottom of the glass partition. After it set, the spacer gel was cast separately on both sides of the glass partition taking care to make the gel on both sides equal and 1 cm in height. Electrophoresis was carried out as usual after charging each one of the two hemispherical chambers with the fluids to be compared. Only one of the two fluids was tagged with a trace of one per cent aqueous bromophenol blue dye. This immediately indicated any leakage between the chambers as dye appeared in the clear chamber to show an unsatisfactory sealed glass divider.
Comparisons attempted in the split gel technique
1. Normal human serum with serum from a case of Wilson's disease separated in the split gel PAGDE technique and stained by amidoblack B.[2] Innoculum volume of each sample was 10 µl. Current was 3 mA and the running time was 2 hours.
2. Normal human serum with serum from a case of Wilson's disease stained for ceruloplasmin.[6]
3. A comparison of proteins in serum and cerebrospinal fluid.
4. Sonicate of strains of Staphylococcus aureus stained for isoenzymes of lactate dehydro-genase[3] and compared with normal human serum.
[Fig. 2a] shows the results of a simultaneous split gel separation of normal serum and a case of Wilson's disease. Ceruloplasmin, a copper oxidase enzyme was demonstrated with special staining, procedure using p-Phenelynediamine, [6] hence is was not necessory to employ a pure ceruloplasmin for comparison. The arrow indicates the total absence of ceruloplasmin in the disease state. Because of the direct comparison the interpretation can be made with confidence. Fig. 2b shows a similar separation stained for ceruloplasmin. Note the total absence of any stainable protein in the lower half of the split gel which is from the case of Wilson's disease.
Similarly, a comparison of cerebrospinal fluid with human serum showed that the former has 4-5 components, while the latter has as many as 22 components when the innoculum was 5 µl each. But with 50 µl of innoculum of cerebrospinal fluid as many as 20 components could be demonstrated. It was easy to identify in the cerebrospinal fluid, by comparison with normal human serum, the major components as IgG, transferrin, ceruloplasmin and albumin. Also a comparison of lactate dehydrogenase (LDH) isoenzymes of Staphylococcus aureus was made with confidence and it was noted that the three LDH isoenzymes of the organism were different from the five of the human serum.
During electrophoretic separation with the split gel, the bromophenol blue marker (charged in only one of the two chambers) was seen to move as a hemisphere. This indicated that there was no mixing of the two fluids being compared. In this technique the components in the two halves travel discretely, without any mixing, in their relative segments of the gel.
Before finalising the method, a number of different partition materials were tried. They included thick waxed paper, plastic (perspex) pieces and formica. However, glass stuck with "Araldite" was the best. We attempted the technique of Spencer and Guest[4],[5] of inserting polysterine partition in the tube after the gel was cast. But it was difficult to get leakproof chambers.
| 1. | Clarke, J. T.: Simplified "disc" (polyacrylamide gel) electrophoresis. Ann. N. Y. Acad. Sc., 121: 428-436, 1964.  |
| 2. | Davis, B. J.: Disc electrophoresis, method and application to human serum proteins. Ann. N. Y. Acad. Sci., 121: 404-427, 1964. |
| 3. | Dietz, A. A., Lubrano, T. and Rubinstein, H. M.: Disc electrophoresis of lactate dehydrogenase isoenzymes. Clin. Chim. Acta, 27: 225-232, 1970. |
| 4. | Spencer, M. E. and Guest, J. R.: Proteins of the inner membrane of Escherichia coli: Identification of succinate dehydrogenase by polyacrylamide gel electrophoresis with sdh amber mutants. J. Bacteriol., 117: 947-953, 1974. |
| 5. | Spencer, M. E. and Guest, J. R.: Proteins of inner membrane of Escherichia coli: Changes in composition associated with anaerobic growth and fumarate reductase amber mutation. J. Bacteriol, 117: 954-959, 1974. |
| 6. | Uriel, J., Gotz, H. and Grabar, P.: Etude de la Ceruloplasmie du serum humain par I'electrophorese en gelose et I'analyse immuno-electrophoretique; Microdetection color imitique du cuivre lieaux proteines [Study of caeruloplasmin in human serum by agar electrophoresis and immuno-electrophoretic analysis; colorimetric microdetection of protein bound copper]. Schweiz Med. Wsch., 87: 431434. 1857. In, "Laboratory Notes For Medical Diagnostics". Editors: B. Bosel. H. Geering and H. Neumann, Published by Behringwerke AG, Germany, 1964. p. 22. |
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