Separation Of Proteins By Focusing Electrochromatography

Focusing electrochromatography was developed based on chromatofocusing. During focusing electrochromatography, a voltage is applied on both ends of column. In this condition, protein molecules are affected by ion exchange and electric field, which can be separated according to the difference of their isoelectric points.The thesis investigated the separation of proteins by focusing electrochromatography, including separation of human serum proteins, separation of the mixture of bovine serum albumin and bovine hemoglobin, separation of the mixture of bovine serum albumin, bovine hemoglobin, ribonuclease and lysozyme, separation of papain. The separation effect of proteins was examined with SDS-polyacrylamide gel electrophoresis. Protein content was determined with Bradford method and protein recovery was calculated. Experimental results showed the application value of focusing electrochromatography on separation of protein.The separation medium can be DEAE-Sephadex A-50 or carboxymethyl cellulose CM-52. The latter is not easy to be blocked in column, so its application range is wider. The mobile phase is buffer with pH change, which forms pH gradient in column.During the application of electric field, focusing electrochromatography obtained better separation of protein mixture than chromatofousing with no electric field. In focusing electrochromatography, the range of pH change covers 6 units of pH and the difference of isoelectric point of separated proteins also includes 6 units of pH. It proved that focusing electrochromatography is suitable to separation of the proteins with large difference of isoelectric points. The recovery yields of proteins were all over 80%.These results indicated that focusing electrochromatography is a novel method for the separation of proteins.