I am trying to purify a non-enveloped virus. The virus is fractionated at high speed centrifugation, so I had to give up purification with CsCl gradient. I have tried Ion exchange chromatography without any luck. I have now been suggested to try a Percoll gradient for purification. In the handbook from GE healthcare, they write that with a discontinous Percoll gradient you can use relatively gentle conditions - such as 400 x g for 15-20 min for centrifugation of your sample.
I haven't been able to find a protocol purifying viruses at low speed centrifugation using a Percoll gradient. If anybody has a protocol or have any experience with this I would like some tips and tricks.
1) In Ultracentrifuge Tubes (40 ml volume) add 22 ml of a sterile solution of Sucrose 30% and the add 15 ml of the supernatant containing the virus in the top of the sucrose solution, avoiding any mixing of the phases.
2) Be aware that you have to fill the six tubes of the ultracentrifuge with the same solution (instead of the supernatant of virus, add PBS in the balancing tubes).
3) Centrifuge at 25.000 RPM for 2hs 4 C in a SW32Ti rotor.
4) Set up the acceleration and deceleration in 1 to avoid any mixing of the gradient. Press Enter and Start.
5) Take out the supernatant and resuspend the virus in 1ml PBS and freeze at -80 C.
It works very nice for me
Interesting question!
At what density these non enveloped VLPs can fractionate?
@ Mauro: Do you concentrate the virus supernatant with a centrifugal concentrator before loading unto the gradient? I normally do this because my virus concentration is so low. Also, I have you tried lower centrifugation than 25.000RPM? and last, when you say take out the supernatant, the virus will not form a band I can extract? do you mean the virus will form a pellet?
@ Ananda: I don't know at what density my VLPs fractionate, that I will have to discover.
Thanks for the help
Hi,
I think Mauro was explained the classical way of concentrating enveloped virus. Concentration of virus supernatant in a centrifugal concentrator is not necessary as sucrose/percoll centrifugation will make a concentrated viral pellet(mostly invisible!). I am sure you checked the cut off value of the filter.
I would suggest you to start with the available material and the protocol suggested for enveloped viruses (I overlay the virus supernatant in 2ml of 20% sucrose solution and run in a swinging bucket rotors for 2h at 39000 rpm, after run discard the liquids by decanting, let the tube dry for few minutes and then I dissolve the pellets in the tip of the tube'sbottom by any buffer of interest).
If it doesnt work why not to try gradients?
Good luck,
Anand
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