how to concentrate/purify virus without ultracentrifugation? - (Mar/29/2009 )
How can one concentrate/purify virus without using ultracentrifugation? I am looking at ultrafiltration/microconcentrator protocols. Does anybody here have an experience in these experiments? What reagents/supplies are needed?
You can use PEG to precipitate your virus. Here is a protocol. see the attached file
http://www.protocol-online.org/biology-for...posts/5721.html
Some companies also sell PEG as a kit. There is a kit called PEG-it.
If there are antibodies against this virus you could try to immunoprecipitate it.
three on Mar 29 2009, 11:14 AM said:
Millipore has filter devices that could help you to concentrate (but not necessarily purify) your viruses. They are meant to concentrate proteins, but the filters could also be used for viruses because the pore size is small enough so that they won't go through the filter. Here is a link for the ultrafiltration devices:
http://www.millipore.com/publications.nsf/.../tp0040en00.pdf
Unfortunately I haven't been very successful with PEG precipitation....so I can't say too much about it. I have used the tangential flow system from Millipore, which work very well for me (but that might be took big for your purposes).....the filters might do. Good luck!
I'm working with Influenza. Do you think that concentration through PEG or the Millipore filters will reduce the infectivity of the virus? For PEG, what MW do you recommend? For the filters, what can be the NMWC? Thanks!
three on Mar 30 2009, 09:02 PM said:
There are several protocols for PEG precipitation, which suggest to use either PEG8000 or PEG6000. Sambrook uses PEG8000 for Lambda precipitation. In regards to the Millipore filters: there are meant for proteins, so the pore-size is very small and it almost doesn't matter which one you would use for viruses because they are much bigger. We used 10kDa and 30kDa in the past.
I have never worked with Influenza and therefore can't tell you if it reduces the infectivity. It might, but I don't think it will reduce it completely (but I can't say that for sure). Maybe there are some papers out there specifically for Influenza where people used similar methods.