7 replies to this topic

[yenchen]

there are many method have been used for purification of virus particle.
1. ultra high speed centrifugation with sucrose, Cscl.
2. low speed centrigugation by 100K cut off membrance.filtration method
3. ion exchange column to absorb virus particle and elution.
4. gel filtration to elute viru particles in fraction.

which method and product can produce good infectious virus particle for the following experiment?

[Curtis]

Definitely ultracentrifugation with sucrose gradient. That's the safest method. After centrifugation you can actually see the band of virus with naked eye. You are sure that's it's only one type of virus, because different viruses have different weights and they move down the gradient according to their weight. after you extract the band you just need to wash it with cold PBS or TEN buffer to remove sucrose, so you don't harm or break the particles by pressure.

[yenchen]

yep, you are right. the sucrose ultracentifugation is good method.
is other method cause some problem in the pcocess of purification?
Because the membrane pore size cut off method and ion exchange method sounds fast and convienet.

[leelee]

This really depends on what you are extracting virus from, what virus you are working with, what you want to use it for and what you have available to you in your lab.

I use none of the ones you have listed!

Edited by leelee, 29 October 2012 - 08:15 PM.

[Curtis]

yenchen, on 29 October 2012 - 08:01 PM, said:

yep, you are right. the sucrose ultracentifugation is good method.
is other method cause some problem in the pcocess of purification?
Because the membrane pore size cut off method and ion exchange method sounds fast and convienet.

ultracentrifuge method takes 2-3 days. but as leelee said the method you want to use depends on the type of the virus and your application too. some of my labmates just extract the alantoic fluid and save it at 4C. They use the virus in PCR and animal studies. But we purify the virus from alantoic fluid so we can save it at -80C for a longer time. We do plaque and HA assay after that and do cloning. So it really depends.

[yenchen]

I work in hepatitis B virus.
if i can't use ultracentrifugation.
I know more purification step and ion buffer system may damage viral particles.
which method is good for my purpose in the purification and " maintain the infectious activity" in my study?
how many viral particles/ per cell could effectively infect human primary hepatocyte cells or natural infection in the human liver?

[leelee]

If I were you, I would be searching the hep B literature for this information. There should be some papers published that outline how they grow, isolate and purify their virus stocks. There may even be standard protocols for exactly this.

[yenchen]

thanks leelee and Curtis.