Electrofocusing, development

Antibodies with specificity for specific carbohydrate residues of antigens have been isolated from serum of rabbits immunized with carbohydrate containing antigens. Eighteen such antibodies were purified by affinity chromatography on adsorbents with ligands of carbohydrate residues. Electrofocusing results showed that all the antibodies occurred in multiprotein forms. A number of anti-carbohydrate antibodies have had useful applications. In the future additional advances with anti-carbohydrate antibodies should be made and these can lead to developments of new and improved products and processes. Some of the applications are summarized ... 

Proteomics started with the development of methods for the purification of proteins. Initially, it involved the purification of one protein at a time mostly by column chromatography. Electrofocusing of proteins was a major development in protein purification, which led into development two-dimensional (2D) gel. Two-dimensional gel resulted in the purification of many proteins on one gel for subsequent characterization. The development of mass spectrometry, genomics, and bioinformatics brought a revolution in proteomics. Now, several proteins can be analyzed simultaneously. 

Sherbet and Lakshmi developed a small-size electrofocusing chamber (Moore and Hibbitt, 1975 Sherbet, 1978) made of Perspex (Figure 7). It consists of two vertical chambers and openings at the top that communicate at the bottom where a needle valve can close the entrance. The chamber of 1.4-cm i.d. contains the density gradient (2-1.5% Ficoll plus 1% ampholine), while the small-bore chamber (0.5-cm i.d.) contains an... 

Figure 27-30,331, shows a ftee-flow recycling apparatus developed by M. Bier and Egan, N.B. in 1979 Electrofocus/78, Haglund, H., Westerfall, J.C. and Ball, J.T. Elsevier, Amsterdam). "The basic idea behind recycling in electrophoresis is that solutes do not have to be completely separated in a single pass... 

The Swedish firm Pharmacia contributed to the development of CF by producing special amphoteric buffers (cf., 4.5.3.3) and ion exchangers (cf., 4.5.2.4). These products are described in detail in a brochure [51] available on request, which serves as a laboratory manual for CF. There is no doubt that chromatofocusing is an effective method for ion exchange separation of proteins. However, because it is a new method, we cannot yet evaluate its contribution to the progress in the field of biochemistry. The author hopes that this method will be found as useful as the electrofocusing method. 

Rotating tube apparatus according to Hjert5n (42), developed for free-zone electrophoresis but works also for electrofocusing. 

LKB-Produkter manufactures a gradient mixer specially designed for their electrofocusing column 8100-10. This gradient mixer has been developed and described by Svensson and Pettersson (59). Any other gradient mixer giving linear density gradients can be used, provided it can be adapted for the appropriate liquid volume. 

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. 

Over the last two years, isoelectric focusing has certainly proved its importance as a refined separation technique in biochemistry. Electrofocusing hinges on three main principles the pH-gradient, the medium for stabilization against convection, and the result measuring technique used. The development of these three main principles will decide the future progress of the technique. 

The method of stabilization against convection determines the character of the equipment. Gels, density gradients or zone convection indeed give the equipment different images. Many of the techniques used in electrophoresis for stabilization today have not yet been studied and tested for electrofocusing. Further developments based on those techniques can be expected. 

Electrofocusing as a preparative technique will have a future. The recent development of zone convection is a demonstration of that. 

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