VIRUS PURIFICATION THROUGH SUCROSE GRADIENT

1. Harvest the supernatant of 293-transfected cells.

2. Either proceed directly, or freeze the supernatant at –80°C after filtering them through a 0.45 µm pore
size filter.
3. If frozen, thaw virus at 37°C, then keep on ice
4. Add 5 ml of sucrose buffer* into clear ultracentrifuge tubes (Beckman, 50 UltraClear TM Tubes, Cat #
344058).
5. Add the supernatant carefully without disturbing the sucrose cushion.
6. Add PBS up to aprox 30 ml. Assure all tubes are balanced
7. Spin down at 26K for 90 minutes at 4°C (Beckman rotor SW28).
8. Remove supernatant by gently aspirating with a vacuum pump and leave the tubes inverted on tissue
paper for a couple of minutes.
9. Clean the walls or the tubes with a tissue paper wrapped pipette.
10. Add 600 µl of cold PBS and incubate on ice for 30 minutes. Resuspend by gentle pipetting.
11. Wash the walls of the tubes with 400 µl of cold PBS.
12. Put fractions together in an eppendorf tube (final volume 1 ml). At this point you may freeze at –
80°C.
13. Spin down in microfuge for eppendorf tube 90 min at maximum speed (4°C).
14. Aspirate the PBS and add 100 µl of cold PBS. Incubate on ice 20 minutes and then resuspend.
15. Wash the walls of the tubes with 50 µl of cold PBS.
16. Put fractions together in an eppendorf tube (final volume 150 µl).
17. Make 3 aliquots of 50 µl and freeze at –80C.
18. Estimate p24 content by ELISA.

*Sucrose Buffer: 20% Sucrose in PBS + 1 mM EDTA (Filter and store at 4C).
Sucrose (FW: 342.30), Fisher Cat # S5-500