I am purifying the virus with the sucrose gradient method, but the result is not good.
1. The virus supernatant was first concentrated by PEG precipitation method.
2. I confirmed the successful concentration of virus by PEG method through the SDS-PAGE and plaque assay. (>50-fold PFU/ml increase)
3. The PEG-concentrated virus was than ultracentrifuged in a
continuous 15–60% sucrose gradient at 110,000g for 18hr at 4 °C,
using a 38.5ml-sized tube.
4. According to the literature, when the ultracentrifugation is over, it is said that the virus band is visible, but no band was visible in my case. So I separated by layers from top to bottom and examined by SDS-PAGE and Western blot to find out which layer the virus appears on.
5. SDS-PAGE and Western blot results showed no target band of virus.
Here I have a question.
Where did the virus disappear during the ultracentrifugation process?
What can I do for trouble-shooting?
Any kinds of tips and advice is welcome.
Have you quantified the virus using Nanodrop or qPCR? I bet that will give you a much better idea.
Maybe this paper could help
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3217642/
Good Luck
Hi,
If you are still interested in ultracentrifugation, here is one idea.
You may try to reduce the centrifugation time. Depending on the virus size, the band may be visible after much shorter run times. For phages, it may be enough to run it 30 min (e.g., in 10–30% (w/v) linear sucrose gradient, ~150 000 g) to see a virus band. With a too long run, viruses may just end up on the bottom of a tube. You can start with some minutes to check if you see a band and then continue with increasing the time and checking the presence of a band again at some time points.
For your point (4), have you also resuspended the pellet and checked it? Viruses may be there if the run was indeed too long.
Best regards,
Tatiana
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