ANALYTICAL AND PREPARATIVE ISOELECTRIC FOCUSING IN GEL‐STABILIZED LAYERS

After the development of the synthetic carrier ampholytes for isoelectric focusing,’-* most methodological efforts have concentrated on the anticonvective stabilization of the pH gradient. A density gradient continues to be the most frequently employed stabilization system in analytical and preparative isoelectnc focusing d e s p i t e some obvious limitations of this technique indicated in a number of publications.4-1o Stabilization of the pH gradient by gel media was quickly recognized as a promising alternative to the density gradient technique. Flat bed and disc isoelectric focusing in polyacrylamide gel are being increasingly used,ll thus confirming the advantages claimed for this stabilization system in some early reports.l*-18 Most of the applications of polyacrylamide gel isoelectric focusing have been confined to analytical separations. Considerable improvement of anticonvective stabilization of the pH gradient has been brought about by the introduction of granular gels such as Sephadex or Bio-Gel employed in layers.le-21 Some of the limitations of both the density gradient and of stabilization by continuously polymerized polyacrylamide gels have thus been eliminated. Excellent resolution and reproducibility, simplicity of apparatus and experimental technique, economy of sample and carrier ampholytes, very high capacity, and efficient stabilization of the separated components are some of the advantages of isoelectric focusing in layers of granular gels. Analytical separations of an artificial mixture of proteins by thin layer isoelectric focusing will be described first, and the results will be compared with those obtained by the density gradient technique with the same protein mixture. Some aspects of stabilization by granular gels, such as possible steric hindrance and isoelectric precipitation of proteins insoluble at the isoelectric point, will then be treated. Isoelectric focusing of enzymes in layers of granular gels will be discussed with special emphasis on different detection methods. Finally, small-scale preparative separations of 100-200 mg quantities of proteins and separations scaled up to gram quantities will be described.