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Additional info for Current Protocols in Protein Science
4, using a Dounce homogenizer, to yield a total volume of 200 to 250 ml (160 ml for liver). Retain a small aliquot of this material for determining protein concentration by the biuret procedure (Basic Protocol 2) and assaying GDH activity (Basic Protocol 3). The ammonium sulfate pellet can be resuspended by careful stirring if a Dounce homogenizer is not available. 8. 4, with at least two changes of buffer, using an MWCO 12,000 dialysis membrane. 9. Centrifuge the material from the dialysis bag 30 min at 40,000 × g, 4°C, and retain the supernatants.
It is important to note that in both of the procedures described below, inattention may result in the concentration proceeding until all the fluid has been removed. It is essential to monitor the progress of the procedure and stop when the desired volume has been reached, because attempts to recover enzyme activity from material that has been taken to dryness by either of these procedures will result in significant losses. Ultrafiltration The Amicon ultrafiltration cell uses pressure of nitrogen gas to force the solution through a membrane that retains molecules above a pre-determined size.
The column is now ready for use. Equilibration of Dowex AG 1X2 Dowex AG 1X2 medium is used in Support Protocol 7. Suspend the Dowex AG 1X2 (200 to 400 mesh, Cl− form) in 3 M HCl and pour into a column of 4 cm diameter to give a settled bed volume of 10 cm. Wash the column before use with 1 liter of 3 M HCl, followed by exhaustive washing with at least 20 liters of water, until the pH of the effluent reaches neutrality. 5 column volumes of the appropriate buffer through the packed column before use.