Isoelectric focussing techniques

Electrophoresis is one of many electromigrational separation techniques which include isotachophoresis, immunoelectrophoresis and isoelectric focussing that have been used to separate various species on the basis of their different mobility in an electric field. These techniques can be used not only to achieve separations but also it is possible to identify the ligand bound to the metal. This can be done by comparing the isoelectric points, immunological behaviours, extent of mobilities or step heights of the sample constituents with those of well-characterised standards. A difficulty, however, is in the determination of the metal constituent itself. Except in the case of radioisotopes, the activities of which can be easily measured, non-radioactive elements can be detected only after further separation steps. 

One- and two-dimensional gel electrophoresis (ID- or 2D-GE) is an important tool in the separation and isolation of intact proteins [9], In ID-GE, the proteins are separated in a sodium dodecylsulfate poly(acrylamide) gel (SDS-PAGE). The separation is according to molecular weight. In 2D-GE, the proteins are first separated by isoelectric point (pi, isoelectric focussing, lEF), and next by molecular weight. 2D-GE is considered to be the most powerful tool in protein separation. Nevertheless, the technique suffers from problems it is labour-intensive, analysis time is long, and the reproducibility poor. Furthermore, hydrophobic proteins do not behave well in the first lEF step and tend to form broad bands. 

As the sample constituent will always move towards their respective isoelectric points, it is not crucial where the samples are applied. Isoelectric focussing gives very high resolutions of samples, and it is particularly suitable for separating isoenzymes, as differences between isoelectric points of only 0.01 pH units are sufficient for separation by this technique. 

Zone detection was accomplished with an UV absorption detector which scanned the length of the tube. Kolin( ) described several free zone electrophoresis systems which used flow in serpentine and helical paths to combat thermally driven convection. Catsimpoolas( ) described an instrumental system with scanning detection for following the course of isoelectric focussing in gel-filled tubes. These techniques have not come into widespread use presumably due to their complexity. 

A much better technique of protein purification is that of isoelectric focussing. Use is made of the isoelectric point of a protein. Suppose that the isoelectric point of a protein is 6.0. If we prepare a pH gradient in a column so that the pH varies from 1-10 and place all the proteins in the sample on top of the column and start the current, all the proteins will migrate dependent on the charge that they possess. At the space where pH of the gradient is 6, while other proteins will continue moving, the desired protein will become immobile since it possesses no net charge the pH is isoelectric for it. The method is discussed in reasonable details in Chapter 12. 

This technique was discovered by H. Svensson in Sweden and has a high-resolution powra. A simple comparison would help establish the method s supremacy over other methods while paper electrophoresis resolves plasma proteins into six bands, isoelectric focussing resolves It into atleast 40 bands. 

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