I am currently in the process of purifying influenza virus on a sucrose gradient but still I have low amounts of protein, you can give me suggestions if not, I use OptiPrep™ Density Gradient Medium. If you're working in this protocol, or can you give me same advices.
thank you
HI Randy
yes i propagate viruses in eggs, but my problem in the step of purification
I lost the virus in the purification step by ultracentrifugation, I find not a band of virus particles but by all, and if I get the virus, the lavge stage I lost
Hi Zeineb,
What g you used in centrifugation. We using 2 step of purification. The first one is a low centrifugation. About 1000-1500 g for precipitation of large particles. The second one is ultracentrifugation. We usually using 103 000g for precipitation viruses.
Hi Zeineb,
if it's any use, we recently published a protocol for this here:
http://www.nature.com/protocolexchange/protocols/3315
OptiPrep works, but sucrose should also be fine. A good prep will give a visible band, but even without a clear band you may still have a decent yield of virus. If you can't find a visible band on your gradient one option is to harvest the entire gradient as fractions (pipetting from the top is probably simplest here), separate a small amount of each fraction by SDS-PAGE and silver stain. You should see the proteins from spherical virions running at about 40% sucrose on a 30 - 60% gradient.
One other thing to consider - if your virus produces filamentous virions (unusual in laboratory adapted strains) purification will be a lot harder.
Hope that's some help,
Ed
Thank you
I will inject 250ug/ml/ pigs, but actually I have only 75 ng/ml, please what is the best concentration of ng for immune response of pigs
Help please
how many eggs are you using to grow up your virus? We usually use about 10 trays (300eggs) which will give you several mg of virus.
Would suggest you start with human vaccine dose (15ug) and go from there. Since you have such a small amount of starting material, you may need to reamplify virus and purify again.
Hi Thomas, Can you suggest or give me a rough idea about how many ugs (micrograms) of the virus could present in 1 ml filtered (cleared) cell culture supernatant having TCID50 of 1X10-8/ml ?
Regards.
You will have to concentrate and test by protein assay such as Coomassie Blue or BCA method. You will also need to blank against control buffer. I have only determined virus protein concentration from sucrose gradient purified/concentrated virus generated from at least 300 eggs. this will give you several mg of virus. Reportedly, one egg should give you ~15ug of purified HA protein. I do not know how much protein is in cell culture media; however, I expect it will be less than what is found in allantoic fluid.
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