Purification of adeno-associated virus (AAV) by FPLC using heparin affinity column

1. Equilibrate HiTrap heparin affinity column with 10 ml PBS-MK at 1 ml/min.

2. Load the column with AAV at 0.3 ml/min.

3. Collect the flow-through in a 14-ml tube.

4. Wash column with 5 ml of PBS-MK buffer using a syringe pump at the rate of 0.3 ml/min.

5. Collect the flow-through in a 14-ml tube.

6. Elute AAV from the column with 2.4 ml of 1 M NaCl/PBS-MK buffer.

7. Collect the first 0.4 ml in a microcentrifuge tube. This is the ‘void volume’.

8. After this initial 0.4-ml void volume, collect the following fractions: fraction 1 is 1 ml, fraction 2 is 0.5 ml and fraction 3 is 0.5 ml. The AAV should come off the column in the first 1.5 ml (fractions 1 and 2).

9. Desalt using a HiTrap desalting column. Wash the HiTrap desalting column with 25 ml of PBS in a 30-ml syringe.

Do not exceed a flow rate of 5 ml/min.

10. Prepare the following:
A 3-ml syringe containing 1.5 ml of fractions 1 and 2.
A 3-ml syringe containing 3 ml of PBS.

11. Position the column to collect fractions into microcentrifuge tubes: the first tube for the 1.5 ml of void flow-through, then ‘exchange’ fractions X1–X3 (1 ml each).

12. Load the 1.5 ml of AAV into the 3-ml syringe.

13. Quickly load the 3 ml of PBS in the second 3-ml syringe.

14. The AAV should be in fractions X1 and X2. Pool fractions X1 and X2 and make 10 aliquots of 200 μl each (or other aliquot sizes) in microcentrifuge tubes. Prepare 1:10 and 1:100 dilutions of the AAV stock in TE buffer for determining titer later.

The vector stock and dilutions can be stored at –80 °C indefinitely. If necessary, store stocks in small aliquots and avoid repeated freezing and thawing.

July 25, 2011 at 9:49 am

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