4. Close the stop-cock and add 20 ml of binding buffer.
5. Open the stop cock to a have a flow rate of not more than 0.5 ml/min .
6. Gently pour the swollen ion exchange matrix to obtain a column bed of 20 cm height.
7. Equilibrate the column with 3 bed volumes of the binding buffer.
8. Load 2-3 ml of the protein solution on the column.
9. Allow one column volume of binder buffer to pass through the column.
10. Set up the gradient mixer as shown in figure 24.2.
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11. Connect the gradient mixture outlet to the column and try to achieve a flow rate of ~0.5 – 0.6 ml/min .
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12. Start collecting fractions of 5 ml each as soon as the NaCl gradient is set up.
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13. If the column is to be reused, regenerate it as follows:
- a. Wash the column with 2 volumes of 3 M NaCl to remove any substance that might still be bound to the column.
b. Wash the column with 2-3 volumes of binding buffer containing 200 ppm sodium azide. Sodium azide prevents microbial growth in the column.
c. Store the column at 4°C.
Figure 24.2: A gradient mixer kept on the magnetic stirrer