I try to purify virus-like particle that express in recombinant yeast.
By sucrose cushion, then subject to equlibirum density CsCl centrifugation.
After I culture yeast for 100mL, harvest, break cell by beat beater, then clarify the lysate by centrifuge at 11,000 rpm for 10 min. Treat Dnase and Rnase, 37 C, 30 min. Then, 14,000 rpm for 20 min. Finally, use 0.45 um membrane filter.
For ultracentrifuge. I layered the lysate on 40%w/v sucrose in PBS buff. Centrifuge 27,000 rpm, 2 hours.
I confuse with the result (already attach image file below). I see a lot of white pellet in the phase of sucrose (middle), and very little pellette in the bottom of the tube. Then I try to suck all solution out, but after the tube is empty from solution, I can not see the pellette in the bottom any more.
The questions:
1. The pellette in the bottom (very little amount) is my virus particle or not?
2. The bulk pellete in the middle layer is the cell debris or not?
3. Should I treat Dnase and Rnase, It will have a bad things with my expermient or not? Normally, I see on paper, no one treat its.
Any suggestion, please provided
Thank you.
Hi,
Treatment with DNAse RNase may not be required,
Viruses /VLPs differ in their density based on which you need to decide on the sucrose % of cushion.
If your VLP is denser than the cushion you used the pellet you are observing contains your VLP.
All other proteins cellular fragments whose density is less than the cushion density are observed as layer over the cushion.
Finally, if your cushion is less denser than VLP all your observations are correct
Hope the information is useful
Dear Rao,
You mean the density of real virus, and the VLP have a difference in desity? Because the real virus have genomic etc., but the VLP have only capsid. So, may be difference in desity?
Thank you
Hi, you can give a try with this protocole.
Host-Associated Metagenomics: A Guide to Generating Infectious RNA Viromes.
Temmam S, Monteil-Bouchard S, Robert C, Pascalis H, Michelle C, Jardot P, Charrel R, Raoult D, Desnues C. PLoS One. 2015 Oct 2;10(10):e0139810. doi: 10.1371/journal.pone.0139810. eCollection 2015.
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