To develop an isoelectric focusing apparatus using a cellulose acetate membrane (Separax EF), we have designed a thermoelectric cooling isoelectric apparatus. This apparatus has two characteristics. Firstly, the cooling system was switched to a thermoelectric cooling system from an ice-cooling system. Secondly, the chamber lid of the electrophoretic apparatus was also devised so that samples could be applied without opening the chamber lid. With this apparatus we could perform the isoelectric focusing without worrying about room temperature and humidity in the laboratory. Applying 2000 V for an extra 5 min with our module cooling system, we achieved a much higher degree of resolution with three sheets of cellulose acetate membrane (Separax EF) overlaid for simultaneous electrophoresis. Thus, three types of information could be obtained from only one electrophoretic procedure.
An apparatus and the procedure for cellulose acetate isoelectric focusing were refined and optimized for routine use in clinical laboratories. The space in the electrophoretic chamber was minimized to maintain high humidity during the run. The water content of the thin cellulose acetate membranes in the chamber was well controlled during isoelectric focusing and the effect of atmospheric carbon dioxide was also excluded. The temperature of the membranes was kept below 2 degrees C, even under conditions of high electric field strength for faster isoelectric focusing of high-molecular-weight proteins. No special training was required for technicians in clinical laboratories to obtain reproducible isoelectric profiles of human serum proteins.
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