I am trying to purify zika virus from cultured vero cells.
Since 2% of FBS was added to the culture medium, I need to remove FBS from the virus supernatant.
For this work, I am using sucrose gradient ultracentrifugation to get purified virus, but it doesn't work well. (I couldn't get any virus band after centrifugation at 100,000xg for 18hrs)
And I've read some papers which used sucrose cushion method to purify specific virus.
The experimental method mentioned in the paper is as follows.
1. Collect supernatant from cells infected by virus
2. Clarify supernatant by centrifugation at 3,200 xg for 10 min
3. The clarified supernatant was then layered over 20% sucrose (w/v) solution prepared in nanopure water in ultracentrifuge tubes on ice.
4. Ultracentrifuge at 110,000 xg for 3 hrs at 4C.
5. Carefully remove the supernatant and invert the tube to drip-dry for 20 minutes at room temperature.
6. Re-suspend pellets with appropriate buffer solution.
7. Centrifuge the viral suspension at 16,000 xg for 10 min at room temperature.
8. Re-suspend pellets with appropriate buffer solution and store at -70C.
Here is my question
1. What is difference between the sucrose gradient method and the cushion method? (I think the cushion method is not perfect enough to purify my virus, because other proteins derived from cells or FBS could be pelleted together with the virus)
2. What about FBS when I perform sucrose cushion method? Does FBS sink with virus if I use 20% sucrose?
Hello!
1. The obvious advantage of a gradient is that you separate your virus from both lighter and heavier fractions. This is especially true when you use a buffer that promotes separation to avoid aggregates. Especially long runs should result in a very narrow localisation of the virus in the gradient. I would always use a gradient for purification and a cushion only for pelleting/ increasing concentration. Consider adapting your buffers, g force and/or sucrose concentration or the method of pumping the gradient.
2. Consider that FBS is a mixture of all types of different proteins and aggregates in various sizes, including oligomers, which pellet easily. I read somewhere that some FBS batches can have sizes up to 800kDa, which would also pellet at 110,000 x g. Therefore you will certainly have some FBS protein contamination if you choose the sucrose cushion method, plus whatever large complexes you have from the cells. The question is if this would interfere with your subsequent assays, or if you can live with some foreign proteins in your virus sample.
3. There are serum-free media supplements available, which would solve your problem right at the source.
I hope this was helpful!
Thank you so much Willi! Your advice would be helpful to me this time. I would try using serum-free medium immediately.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Biomolecules
- Nucleic Acids
- RNA