Hi I am looking for any alternative protocols to purify TMV virions, other than the commonly used PEG precipitation.
Two main problems that I have with PEG protocol is that,
1\ any macromolecule will co-precipitate as well
2\ so that PEG cannot purify full length (300nm) TMV virions from other shorter rods (probably breakage), and other small discs. (thanks to people responded to my earlier question)
So I am wondering if there is any purification protocol or method that can get mostly the full length rods and rid breakage ones and discs?
Thanks a lot in advance
Hello Tommy,
In the past I have used Zinc chloride to ppt bacteriophage, and got very good yield,and fantastic DNA that could be digested by restriction enzymes. I have attached an article from 1991.. works well. Hope it works with TMV too.
Good luck
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC328918/pdf/nar00099-0320.pdf
Hi Tommy,
did you consider purification using a CsCl-gradient? I have used it to purify phages and it worked pretty well. This might help you to avoid contamination due to co-precipitating molecules.
Best,
Christian
you may use differential centrifugation to separate the TMV particles if you know the sedimentation in Swedberg units. Now you spin your suspension at a lower value of S which will clear all unwanted dirt and then spin it in the exact S value for TMV. By this way you can deviate from the PEG method (though that is the most preferred method).
Use CsCl density gradient to separate TMV particles from macro-molecules.
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