Lentivirus Purification Anion Exchange Column

HiTrap Q HP

1. Wash strong anion exchange column HiTrap Q HP (5 ml, Amersham Biosciences) with 25 ml of PBS, pH adjusted to 7.5, flow rate of 5 mL/min.
2. Wash column with 25 ml of PBS-1M NaCl, 5 ml/min.
3. Wash column with 25 ml of PBS, 5 ml/min
4. Mix 0.5 ml concentrated lentivirus with 1 ml starting buffer, spin for 5 min to remove large debris at 6000× g for 5 min.
5. Load sample to column.
6. Wash with 25 ml washing buffer
7. Elute with salt gradient step (0–1 M NaCl) for 10 min, 2.5 mL/min of flow rate, collect 1 ml/tube.
8. Virus peak: about 40 ml
9. Use Centricon to desalt.

1. Transfect 2 x 10⁶ 293T cells with each 8 μg V, G, and pLVX construct
2. After 48 h, the supernatant was harvested and filtered through a 0.45-μm pore size filter.
3. Concentrate to 1/100 volume by ultracentrifugation with 50 000 × g at 4°C for 2.5 h using a SW28 rotor.
4. Resuspend virus particle pellet in PBS and frozen at -80°C until use
5. Wash column with 5 column volumes (25 mL) of starting buffer (PBS adjusted pH at 7.5) at a flow rate of 25 mL/min.
6. Wash column with the elution buffer (PBS, pH 7.5 containing 1 M NaCl) at a flow rate of 25 mL/min.
7. Equilibrate column with the starting buffer at 2.5 mL/min for 10 min.
8. Mix concentrated virus vector (0.5 mL) suspended with 2.5 ml of starting buffer
9. Spin at 6000 × g for 5 min to remove large particles.
10. Load virus suspension to column.
11. Wash with starting buffer.
12. Elute with NaCl gradient (0–1 M NaCl) for 10 min, at 2.5 mL/min.
13. Concentrate the virus using Centricon Plus-20, cut-off 100 000 molecular weight; Millipore, Bedford, MA, USA). The membrane should be pre-sterilized by washing with 70% ethanol