Chromatography electrofocusing

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. 

Purified by Sephadex G-200 filtration and DEAE-cellulose column chromatography. Hexosaminidase A was further purified by DEAE-cellulose column chromatography, followed by an ECTEOLA-cellulose column, Sephadex-200 filtration, electrofocusing and Sephadex G-200 filtration. Hexosaminidase B was purified by a CM-cellulose column, electrofocusing and Sephadex G-200 filtration. [Srivastava et al. 7 Biol Chem 249 2034 1974.]... 

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).

The purification was by chromatography on DEAE cellulose. Electrophoresis of the purified enzyme and the original filtrate are shown in Fig. (27) [71]. Rabbits were immunized with the pure glucoamylase [72]. Antibodies in the serum were purified by affinity chromatography. The affinity pattern for glucoamylase purification is shown in Fig. (28). Electrofocusing and assay by the coupled method for antibodies is shown in Fig. (29). The glucoamylase antibodies consist of 8 isomers [73],... 

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 ... 

All the anti-carbohydrate antibodies purified by affinity chromatography are isomers of a different number of isoforms. The forms can be separated by electrofocusing and all have been found to have combining activity for the immunodeterminant of the same antigen by the method of coupled analysis of electrofocusing and agar diffusion [88], The isoforms of antimonosaccharide antibodies are shown in Fig. (48). 

Both the quantity and properties of cellulases produced by microorganisms depend on the culture conditions. Commonly, cellulases are produced by culture of the organism either (a) in a liquid medium, which may be stationary, shaken, or submerged with aeration, or (b) by a Koji process on a solid substrate such as wheat bran (7). The complexity of the crude cellulosic carbon source usually leads to the production of a mixture of hydrolytic enzymes which may include amylases, proteases, chitinases, etc., in addition to the cellulases. Separation of proteins from culture filtrates by high resolution techniques such as chromatography, electrophoresis, or electrofocusing often reveals a number of enzyme species which may differ in specificity toward cellulosic substrates. These forms may represent ... 

These methods, which include chromatography, electrophoresis, electrofocusing, and some of their applications for the separation of proteins and purification and isolation of stabilized enzyme -substrate compounds, are described and discussed in Sections III and IV. 

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