Lentivirus Purification PEG + Q Membrane by FPLC for in vivo

Concentration of lentiviral vectors using Mustang Q anion exchange chromatography
1. Precipitate lentivirus from 15 cm plates using PEG.

2. Suspend lentivirus in  25 mM Tris-HCl, pH 8.0, 0.1 M NaCl (Buffer A)

3. Load suspension onto a Mustang Q Acrodisc (bed volume 0.18 ml) attached to an AKTA purifier HPLC system using the system's pump at a flow rate of 1 to 10 ml/min.

4. Wash with 10 ml of 25 mM Tris-HCl, pH 8.0, 0.1 M NaCl (Buffer A)

5. Elute lentivirus with a step gradient ranging from 0.1 to 1.5 M NaCl in 25 mM Tris-HCl, pH 8.0 (Buffer B). Collect 1 ml fractions.

6. Pool the eluted fractions showing the highest TU levels, load them onto a 2.0-ml Sepharose CL-4B column (Amersham Pharmacia) equilibrated with Tris-HCl, pH 7.4, and 150 mM NaCl.

7. Spin at 500 x g for 1 min and analyse the excluded volume by RT-PCR lentivirus titering.

8. Concentrate/desalt combined fractions using 100 KDa spin column, concentrate to 2 ml. 

9. Aliquote to 200 ul, print labels, store lentivirus tubes at -80°C.