Hi everyone,
I'm working with Picornaviruses and I'm fairly new to purifying viruses. I've been trying to isolate full and empty capsids in a sucrose gradient for the past 3 weeks but no luck so far. This is what I have done so far:
1) Sucrose linear gradient from 30-60% (w/v) using a gradient maker, 25,700rpm for 20 h at 4 °C.
2) Sucrose step gradient from 30-60% (w/v) with 5% increments (30%, 35%...), 25,700rpm for 20 h at 4 °C.
In both the cases, I found my virus in the pellet and there was no fractionation at all. Ideally, I would like to see empty and full capsids as bands in the gradients as mentioned in the literature.
Not sure what I'm doing wrong here- Any suggestions regarding this would be highly appreciated.
Thanks.
Dear Nadishka Jayawardena
Pls. find the attached files and the references, good luck
Hoping this will be helpful to your project
Regards
Prof. Houda Kawas
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC252724/
https://www.ncbi.nlm.nih.gov/pubmed/3001350
https://www.ncbi.nlm.nih.gov/pubmed/8380179
https://books.google.com/books?id=V0AG27Byy4oC&pg=PA77&lpg=PA77&dq=Picornavirus+purification&source=bl&ots=-2NkCqR_5O&sig=W3J6Rup0SXnQjlq3_N61CtnOwLc&hl=ar&sa=X&ved=0ahUKEwi5seKB5oPTAhVGEpoKHeuZA0AQ6AEIZjAI#v=onepage&q=Picornavirus%20purification&f=false
https://books.google.com/books?id=zMg-aBWz8GkC&pg=PA428&lpg=PA428&dq=Picornavirus+purification&source=bl&ots=FNgsK4_656&sig=aeJ9jBbTTciPUXw0R627Hq_M5Cs&hl=ar&sa=X&ved=0ahUKEwio4-uY5oPTAhVJOJoKHSqnCJo4ChDoAQgtMAI#v=onepage&q=Picornavirus%20purification&f=false
http://www.nature.com/nature/journal/v517/n7532/fig_tab/nature13806_SF1.html
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0020005
https://books.google.com/books?id=Eeb7CAAAQBAJ&pg=PA114&lpg=PA114&dq=Picornavirus+purification&source=bl&ots=0WwuCZasie&sig=eCrSVJ8pvnhhosk0zShs_pBYXIo&hl=ar&sa=X&ved=0ahUKEwi5seKB5oPTAhVGEpoKHeuZA0AQ6AEIajAJ#v=onepage&q=Picornavirus%20purification&f=false
https://books.google.com/books?id=Xrk7N8jPZZUC&pg=PA4&lpg=PA4&dq=Picornavirus+purification&source=bl&ots=JqOo6rc7q7&sig=Fq7VaHclBtlqGI6WXlJOnZsxc7I&hl=ar&sa=X&ved=0ahUKEwio4-uY5oPTAhVJOJoKHSqnCJo4ChDoAQhNMAY#v=onepage&q=Picornavirus%20purification&f=false
http://pqdtopen.proquest.com/doc/1271758646.html?FMT=ABS
Also
Hoping this will be helpful to your project
http://www.bio-protocol.org/e1117
https://www.researchgate.net/post/Sucrose_gradient_for_Picornavirus_purification#58dfe746f7b67eb3d3515832
https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=16&cad=rja&uact=8&ved=0ahUKEwio4-uY5oPTAhVJOJoKHSqnCJo4ChAWCEcwBQ&url=https%3A%2F%2Fspiral.imperial.ac.uk%2Fbitstream%2F10044%2F1%2F30829%2F1%2FNewman-J-2015-PhD-Thesis.pdf&usg=AFQjCNGTbak0HTBMT0XkiQJfIAKvveIu6Q&bvm=bv.151325232,d.bGs
75
Hi Nadishka
You can read this paper1
https://jvi.asm.org/content/57/1/275.full.pdf
4
https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0020005
7
https://escholarshare.drake.edu/bitstream/handle/2092/532/dt1991mjs001.pdf?sequence=1
10
http://www.microbiologyresearch.org/docserver/fulltext/jgv/65/1/JV0650010129.pdf?expires=1491254866&id=id&accname=guest&checksum=21DCBA904B9CBC39C1D928407AC60754
11
http://www.microbiologyresearch.org/docserver/fulltext/jgv/72/11/JV0720112721.pdf?expires=1491254884&id=id&accname=guest&checksum=C0B2D1311CBAD1F95D20C761E7F1F725
12
http://www.uta.fi/med/pevnet/news/B12%20P3%20Antiviral%20Res.%202014%20Apr;%20104;%2093-101%20TK,%20OL.pdf
Thank you very much for your valuable insights, Prof. Kawas and Laith Al-Ani!
Hi Clinton, our lab has previously used the CsCl gradient but didn't have much luck with separating two separate fractions containing full and empty capsids. It was rather a fraction containing mostly full capsids and a little bit of empty capsids. Thank you!
Hi Devendra, concentrating the virus with PEG is another step that I would like to add. Have you done any experiments with and without PEG to see if there was any difference? Thanks.
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