Measurement after Electrophoretic Separation

Abstract

This chapter focuses on electrophoretic separation of human α-amylase that has been carried out on paper, agar, polyacrylamide, and cellulose acetate. It is applied in clinical chemistry. The electrophoretic separation is carried out on a slide covered with a layer of agar, which is then laid on a translucent starch substrate. Optimum α-amylase activity is measured at 20 mM Ca 2+ and 140 mM Cl − ; absence of phosphate prevents phosphorolytic degradation of starch. The whole 24-hr urine should be collected in a glass bottle containing 1 ml toluene. Urine should be sterilized by passing first through a 1.2 μ Millipore ® filter and then through a 0.12 μ filter. Under toluene the isoamylases are stable for weeks at 4°C. Insert vertically two rectangular strips of filter paper 6 mm wide immediately adjacent to one another in the agar on the slides, 1 cm from the middle line on the anode side. After the electrophoresis either remove the agar from the slide and carefully lay on a substrate plate, or use a cellulose acetate carrier. Remove agar or cellulose acetate strips and pour the iodine solution over the Petri dish. If the hydrolysis of the starch is observed by eye, then method is specific for α-amylases.