Disc electrophoresis techniques

Matoltsy, A. G, and Matoltsy, M. N., A study of soluble proteins of nomial and pathologic horny tissues by a modified disc electrophoresis technique. J. Invest. 

Disc Electrophoresis. Resolution in zone electrophoresis depends critically on getting sample components to migrate in a focused band, thus some techniques ate employed to concentrate the sample as it migrates through the gel. The most common technique is referred to as discontinuous pH or disc electrophoresis. Disc electrophoresis employs a two-gel system, where the properties of the two gels are different. 

Disc electrophoresis was first iatroduced ia the early 1960s (11—13) as various techniques using polyacrylamide gels were being explored and designed. Original work employed several buffer systems and different polyacrylamide gels in order to first concentrate and then separate compounds (14). 

The Laemmli SDS-PAGE protocol is one of the most important analytical techniques in analytical protein separation. It is a system with discontinuous pH gradient (disc electrophoresis) and consists of a stacking and a separation gel different in acrylamide concentration and pH. The separation gel may be formed with homogenous acrylamide concentration or with an increasing gradient. 

Maurer, H. R. In Disc Electrophoresis and Related Techniques of Poly-... 

Discontinuous zonal electrophoresis, known as disc-electrophoresis for short, is the electrophoretic technique that is most often used in protein analysis. The method, originally developed by Omstein and Davis, has given rise to several derivative techniques, notably the well-known SDS-PAGE method of Laemmli (1970). The technique is thoroughly covered in the literature, reflecting its importance in protein... 

Proteins can ba fractionated by electrophoretic techniques on the basis of one or a combination of their three major properties size, net charge and relative hydrophobicity. Electrophoresis under native conditions is ideal for soluble proteins, where biological properties can often be retained. In contrast, more vigorous and often denaturing conditions must be used for analysis of less soluble proteins. Electrophoretic separations can be carried out using either a continuous or discontinuous (Multiphasic) buffer system. The techniques are referred to as continuous zone electrophoresis (CZE) or discontinuous ("disc") electrophoresis (also known as multiphasic zone electrophoresis, MZE). 

By a suitable combination of some fundamental electrophoretic techniques their advantages can be exploited for better separations. For instance, in discontinuous electrophoresis (disc-electrophoresis), isotachophoretic arrangement is utilized in the first part of the experiment in order to concentrate the sample components, and to arrange them according to their effective mobilities. In the second part of the experiment individual zones are separated on the principles of zone electrophoresis. In other cases combinations of electromigration and other principles (for instance, immunoelectrophoresis) are exploited. 

Maurer, H.R. (1971) Disc Electrophoresis and Related Techniques of Polyacrylamide Gel Electrophoresis, Walter de Gruyter, Berlin, New York. 

A few practical notes should be emphasized regarding SDS electrophoresis. Potassium salts should be avoided and sodium salts used in their places because potassium dodecyl sulfate is quite insoluble. In addition, even sodium dodecyl sulfate is insoluble below about 10°C. Although not specifically cited above, disc gel electrophoresis may also be used in the presence of SDS. These techniques, described by Studier (11) and Ames (12), are of great advantage when the sample volume is in excess of 10 to 20 /Ltliters per gel. A widely used buffer system for SDS acrylamide gel electrophoresis is that developed by Laemmli (41). 

The original technique developed by Weber and Osbron [94] exists today in two variations with regard to the spatial arrangement in the form of disc (discontinuous) electrophoresis on gel rods, or in the form of slab gel separation. 

In zone electrophoretic techniques described so far, even if all the care Is taken to layer the sample over the gel, the sample can never be loaded In a sharp band and this diffused layer of sample Impedes In a sharp resolution of the components. Disc gel electrophoresis (so called because of the discontinuous buffer employed and discoid appearance of the macromolecular zones) is a modification of conventional zone electrophoresis, which allows the sample to enter the gel as a sharp band, thereby helping further resolution. Here the macromolecule mixture to be analyzed Is subjected to an electric field in a retarding gel support that is separated Into two sections differing In porosity and buffered at different pHs. The macromolecular mixture migrates from the more porous into the less porous gel, a process accompanied by a change In pH. As a result, each macromolecular species becomes concentrated into a very thin, sheirp band, producing much higher resolution than can be achieved In a continuous buffer. 

Dipeptidyl Carboxypeptidases.—Dipeptidyl carboxypeptidase (angiotensin I-converting enzyme) has been purified by conventional techniques from a particulate fraction of porcine kidney cortex. The purified enzyme gave two protein bands on standard disc-gel electrophoresis, but showed a single protein component after treatment with neuraminidase. It was concluded that the preparation was a mixture of sialo- and asialo-enzymes and that neuraminic acid residues did not contribute to the catalytic activity. 

---