Gel electrofocusing

Isoelectric focusing has undei ne extensive developments in recent years. The most important are self-stabilizing zone convection electro-focusing according to Valmet (40), and the relatively new branch of gel electrofocusing. 

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Figure 4. Gel electrofocusing of T. koningii cellulose (A, left), and Ct (B, right) in an ampholyte covering the pH range 3,5-5.6. Ct was isolated as in Figure 1 and purified by further chromatography on DEAE-Sephaaex (6).

Fig. 15.—Gel electrofocusing of anti-fucose and anti-BSA antibodies gel B = anti-BSA antibodies, gel F = anti-fucose antibodies gel E = embedded gel of anti-fucose antibodies P = precipitin band, and T = solution of fucose-BSA. (Reprinted with permission from Journal of Protein Chemistry, Volume 13, J. H. Pazur, B. Liu, and T. F. Witham, pp. 59-66, copyright 1994 Journal of Protein Chemistry.)...

Fig. 30.—Gel electrofocusing of purified myeloma proteins. G = gel stained for protein E = embedded gel P = precipitin area A = antigen solution. (Reprinted from Immunology Letters, Volume 5, J. H. Pazur, M. E. Tay, S. E. Rovnak, and B. A. Pazur, pp. 285-291, copyright 1982 with kind permission of Elsevier Science—NL, Sara Burgerhartstraat 25,1055 KV Amsterdam, The Netherlands.)...

E4. Ernes, A. V., Latner, A. L., Rahbani-Nobar, M., and Tan, B. H. A., The separation of plasma lipoproteins using gel electrofocusing and polyacrylamide gradient gel electrophoresis. Clin, Chim. Acta 71, 293-301 (1976). 

Isoelectric focusing and electrofocusing are names that have been accepted, and in current use, since 1967. Before that time the technique was called isoelectric separation, isoelectric fractionation, isoelectric condensation, isoelectric analysis and focusing electrophoresis, as well as stationary electrolysis. Terms such as density gradient electrofocusing or gel electrofocusing indicate the medium in which the experiments are carried out. 

The stabilization techniques of importance today in electrofocusing are (a) density gradient electrofocusing, (b) gel electrofocusing, and (c) zone convection electrofocusing. These methods are described in detail later in this article. 

Hitherto, gel electrofocusing has been carried out in thin layers and troughs, or else in narrow tubes. Very thin layers have been used by Awdeh et cU. (39), as well as by Catsimpoolas (28). Leaback and Rutter (37) used a special trough for casting slabs of polyacrylamide. The latter method permits a certain amount of preparative work. Riley and Coleman (30) also used thin layers. 

There is another factor which helps to make gel electrofocusing simpler than gel electrophoresis. The profile of the separation is not affected by the structure of the gel. However, if the gel has small pores, it may slow down the over-all time of separation, and perhaps stop very large protein molecules altogether. 

In gel electrofocusing, however, no matter how each protein component is distributed at the beginning of a run, it always ends up at the point on the gradient where the pH is equal to the isoelectric point of the protein component. It becomes sharply concentrated there and stays there. In other words, the final position of each protein component is independent of the gel pores. This can be strikingly demonstrated by parallel tests, applying the sample at different places on the gel. After electro-focusing, it will be found that the same component gathers in the same place. If this should not be the case, the pores of the gel are too small for the protein molecules to move. 

If it is desired to make a closer study of a protein component after it has been electrofocused, a small piece of the gel can be cut out. The piece should be soaked in water and the extract should be measured. This technique has been employed by Awdeh and co-workers, Wrigley, and others. However, sucrose density gradient electrofocusing is probably preferable if more exact studies are necessary. Gel electrofocusing is cheaper and simpler. Thus it can serve as an excellent pilot for density electrofocusing, which is relatively expensive and time-consuming. 

Photo-polymerization is often the most convenient way, but it has certain limitations. It can only be used for the pH range 5-8 or for the whole pH range 3-10. Chemical polymerization can be used for all pH ranges (29). It is recommended that the protein should not be applied immediately after polymerization. This should be allowed to finish overnight or some comparable period. It appears that many tricks known in the fields of gel electrophoresis and disc electrophoresis for polymerization and staining can also be applied to gel electrofocusing. The main special problem of gel electrofocusing is how to stain protein zones without disturbance by the carrier ampholytes. 

Figure 23. The gel electrofocusing apparatus used by Leaback and Rutter (78). It comprises a perspex lid bearing terminals connected to horizontal carbon or platinum electrodes (E), and a glass base to carry the gel (G) and the electrode wells (A and B). The base consists of a glass plate to which 4 strips of ass are bonded (with Araldite epoxy-resin) to form a watertight well 18 X 8 X 0.2 cm, a perspex strip 8 X 2 X 0.2 cm displaced at (A) and another at (B) before the well is filled with polymerization mixture. Electrode wells (A) and (B) are filled with aqueous solution of phosphoric acid and diaminoethane respectively. The samples are added to slots (S) and with the lid in position, a potential difference is applied to the electrodes with the current kept at or below 2 mA. A moist pad (C) prevents excessive evaporation from the gel surface. (Leaback and Rutter, 37.)...

Figure 24. Gel electrofocusing according to Fawcett. Ampholine pH 7-10, anode at top. From left to right HbA, HbA+S, Hb A+C, whale myoglobin, horse myoglobin, mixture of whale and horse myoglobin, HbA. (Fawcett, FEBS Letters 31.)...

Wrigley points out the possibilities of combining gel electrophoresis with gel electrofocusing. In a private communication to him in 1968, Margolis and Kenrick (69) described how they had followed up the first fractionation by gel electrofocusing. The gel was not dyed but was laid directly on a fresh slab of gel for electrophoresis in a second dimension to achieve better separation. They used a polyacrylamide gel with gradually diminishing pore size in order to obtain increased resolution due to molecular size. 

Riley and Coleman (30) have also presented work on immunoelectrofocusing. They used 1.5% agarose ( Seakem, Bausch and Lomb) on a microscope slide in the traditional way, except that the conventional buffers were replaced by 2% Ampholine solution, pH range 3-10. The anolyte and catholyte were the same as for gel electrofocusing in the polyacrylamide system, that is, 1% phosphoric acid and 2% ethylene diamine. Riley and Coleman did their immunoelectrofocusing on human serum. With agargel a certain degree of electroendosmosis is obtained which can influence the result. 

The technique begins with gel electrofocusing in tubes, in a similar way to the method of Wrigley (29) or Riley and Coleman (30). After focusing, the gel is removed from the tube and applied to a thin layer gel. 

Routine analytical applications of the fingerprint type will be widely used, especially in clinical chemistry. Gel electrofocusing equipment will without doubt soon be available, provided with arrangements for easy handling and automated applications. 

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