By Willis A. Wood
The severely acclaimed laboratory normal, Methods in Enzymology, is among the such a lot hugely revered guides within the box of biochemistry. seeing that 1955, every one quantity has been eagerly awaited, often consulted, and praised through researchers and reviewers alike. The sequence includes a lot fabric nonetheless suitable at the present time - really an important book for researchers in all fields of existence sciences.
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Additional info for Carbohydrate Metabolism, Part C
Inclusion of EDTA, fl-SH EtOH, and glucose are all necessary to preserve maximum enzyme activity. KC1 was not ineluded in the chambers or gel since the presence of KCi destroyed the voltage-current relations during the eleetrophoresis. After eleetrophoresis the gel was cut into a 10 em X 10 em square, care being taken to include all the areas where glueokinase should be located. This depended on the slots to which the sample was applied and the migration of the enzyme (usually about 9 cm under these conditions).
Inclusion of glucose prevented large losses of enzyme activity during the (NH4)2S04 fractionation. To each 100 ml of supernatant from step 1, 85 ml of saturated (NH,)2SO~ solution were added with constant magnetic stirring to give 48% saturation. After 15 min, the precipitate was spun down at 9000 rpm in the GSA head in the Sorvall centrifuge. To each 100 ml of the resulting clear red supernatant was added 60 ml of saturated (NH4)2SO~ solution to give 68% saturation. The precipitate was spun down and stored at --20 °.
The dialyzed protein solution was adsorbed on a DEAE-cellulose colmnn (160 ml) previously equilibrated with the dialysis buffer. 4 M KC1 dissolved in the same buffer was applied thereafter at a flow rate of 20 ml per hour. 24 M KC1. 5 containing 2 mM 2-mercaptoethanol, 2 mM EDTA, and 5 mM glucose. The enzyme solution was dialyzed overnight against this buffer. Step 5. First DEAE-Sephadex A-50 Chromatography. The dialyzate was adsorbed on a DEAE-Sephadex A-50 column equilibrated with the Tris dialysis buffer.
Carbohydrate Metabolism, Part C by Willis A. Wood