I would like to concentrate and purify FMD virus preparation (to get rid of non-structural proteins of less than 100 KDa) . My idea is to initially concentrate the virus by PEG precipitation, then use this preparation for further purification and then concentration by adapting a methodology, which members of RG suggested.
The manufacturers of amicon spin filters state that for a protein to pass through the filter it has to be 1/5 of the MW cut off. So you would need a 500k NMWCO filter to efficiently filter out 100K proteins. However, my experience is that this is optimistic at best. I have attempted to filter histone H4 14kDa through a 100K NMWCO filter and almost no histone H4 filters through. Using SEC as suggested by Joachim is the best choice. Try to find a resin that has a cut off such that 100K or more comes out in the void volume. Collect your protein in the initial fractions and dump everything else then concentrate. Good Luck
Hi,
you could try to dialyze your samples. Sigma sells dialysis membranes with a 100 kDa cut-off (spectra-por float-a-lyzer, Z727253-12EA).
regards,
Bert
Hi,
U can try Viva spin columns ( http://www.vivaproducts.com/downloads/vivaspin-2-500-operating-instructions.pdf) .
regards
Jegathes
If your proteins are monomeric and do not associate non specifically, then any one of the above suggestion should work. There are several products in the market and i am not certain if you get 100% removal. But this suggestion is from biochemist point of view. Virologists could suggest more on this specifically. Good luck
Hi Bert & Jegathes, thanks for your reply. By dialysis, I can get rid of NSPs. Then for concentration I can use Vivaspin columns as Jegathes suggested.
There is also gel-filtration (size-exclusion chromatography), however, centricons (or viva spins whatever you would like to call them...) are easiest. Mind however that there are proteins that like to bind to the filter matrix. If you seem to "magically" lose protein during concentrating, add some detergent (refer to manufacturer´s manual) that is not compatible with the concentrator in order to resolubilize your protein.
Srinivas - the Vivaspin/centricon concentrators have different molecular weight cut-offs. So by choosing one with a high molecular weight cut off, and this step could get rid of the contaminating low MW proteins AND concentrate the virus prep. in one step.
The manufacturers of amicon spin filters state that for a protein to pass through the filter it has to be 1/5 of the MW cut off. So you would need a 500k NMWCO filter to efficiently filter out 100K proteins. However, my experience is that this is optimistic at best. I have attempted to filter histone H4 14kDa through a 100K NMWCO filter and almost no histone H4 filters through. Using SEC as suggested by Joachim is the best choice. Try to find a resin that has a cut off such that 100K or more comes out in the void volume. Collect your protein in the initial fractions and dump everything else then concentrate. Good Luck
For me it sounds like all suggestions are from protein purifying researchers. These are standard techniques we use in the lab. I am not sure if a virus prep could be purified with gel filtration. I am curious about right strategy from virus purifying labs.
In support of Ted and Joachim, I found this following paper with a quick search in Google.
Chromatographic preparation of purified structural proteins from foot-and-mouth disease virus
S. Bernard, Josiane Wantyghem, Jeanne Grosclaude, J. Laporte
Biochemical and Biophysical Research Communications
Volume 58, Issue 3, 4 June 1974, Pages 624–632
Centrifugation using sucrose gradients is an old-fashioned and somewhat tedious (gradient making and fractionation require some practice), but otherwise excellent technique to purify macromolecular complexes (viruses included). The main benefits are excellent separation and absence of losses of the material on the membrane.
For FMD virus, ultracentrifugation through 15-45% sucrose gradient has been reported: Yang et al., Veterinary Immunology and Immunopathology 115 (2007) 126–134. All 100 kDa proteins will remain on top of the gradient, and the virus particles will be in the middle. Note that sucrose gradients can be as small as 15 ml (you need a small gradient mixer, though). After centrifugation, place capillary attached to a peristaltic pump and collect 0.5 ml fractions (drop-wise) from bottom to top; measure OD at 280. The virus is likely to be concentrated in 2-3 fractions.
Size exclusion as well as ion exchange and other capture chromatography methods can be used to purify a variety of viruses. I have used gel filtration S200 (Sepharose, Sephacryl) from GE to purify certain retroviruses and even Herpes simplex virus. They also make larger resins now for iunfluenza size exclusion. Look on the GE web site for additional information about your requirements.
Srinivas, I agree with Maria Kireeva-although a little bit tedious, sucrose gradient or CsCl banding will give you pure virus. The following Methods in Mol. BIol reference might prove useful for CsCl banding. Methods Mol Med. 2007;130:223-35
Good Luck.
you may also consider using Optiprep.
http://www.axis-shield-density-gradient-media.com/virusindexes.htm
far less toxic than CsCl + simple protocols.
Hi,
For our virus purification we will do a PEG precipitation, then CsCl gradient followed by dialysis. Maybe you should look at trying HPLC and monolith columns.
Heather
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