Isoelectric focussing preparative

Isoelectric focussing allows the separation of zwitterionic analytes such as proteins or peptides according to their isoelectric point, pi (section 1.1.1.1). lEF is applied to the separation and purification of proteins, peptides and amino acids on an analytical as well as preparative scale. The pi of a protein depends on the sum of all charges, as well as the 3D structure and post translational modiflcations such as phosphorylation, glycosilation and changes in oxidation state. The pi is, thus, a valuable parameter for studying post-translational modifications of proteins. 

Sphingomyelinase is believed to be a lysosomal enzyme. It has been partially purified from human liver and spleen. Isoelectric focussing of a partially purified preparation of the enzyme has permitted iden-... 

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. 

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