After regular PEG purification, can I use 25% chloroform (no phenol) to further purify the TMV virions from host proteins / free coat proteins? and what about getting rid of some remaining pigments? If so, why is that? thanks
The reason I ask: I saw a paper mentioned (just mention) this as a last step purification of TMV. I dont understand if chloroform can rid host proteins why it wont interrupt TMV?
It would help to see the whole protocol in question. I do this step occasionally to get rid of plant pigments which sometimes make the purified virus rather green. It should also remove membrane proteins which might for some reason co-purify with TMV.
Dear Tommy (also dear Tomas) the question also is of interest to me (only from a methodological standpoint). While I am looking forward too to see / get the reference for the "chloroform as a last step in purification of TMV" mentioning paper I would love to read your acknowleged comment on a "specific" purification instruction for CP-TMV viruses compared to other viruses (ok, it is an old paper, cf. http://www.apsnet.org/publications/phytopathology/backissues/Documents/1983Articles/Phyto73n06_936.pdf
GERGERICH RC, SCOTT HA & FULTON JP (1983): Regurgitant as a determinant of specificity in the transmission of plant viruses by beetles, Phytopathology 73, 936-938
==Ex Text MATERIALS&Methods, VIRUSSES & VIRUS Purification
All viruses were purified by chloroform-butanol extraction followed by two to three high- and low speed centrifugations and resuspension in 0.01M phosphate buffer, pH 7.2. Bean leaves infected with CP-TMV were homogenized in 0.01 M phosphate buffer (pH7.2) containing 0.1mM disodium EDTA. The homogenate was clarified by low speed centrifugation, and the virus was precipitated from the supernatant by the addition of 4% PEG (6000MW) and 0.3 M NaCl. After 30 min of stirring, the virus was pelleted by low-speed centrifugation, resuspened, and purified further by alternate high- and low speed centrifugation.==) (not knowing if this also could be followed for further TEM-visualization)
I greatly appreciate your kind reply, best regards, Wolfgang
Dear Wolfgang you have raised an interesting question. Chloroform or butanol/chloroform clarification is used as a fast way to remove most of the plant components at the early stages of purification. Some plant viruses are more likely to stay in aqueous phase than other. The behavior of a particular virus should be determined empirically. I dont know the significance of particle symetry in this context, but it should be noted that the first two viruses mentioned in the Phytopathology paper (SBMV and BPMV) are spherical while CP-TMV is rod shaped. It is possible that they have simply followed standard purification protocols for each virus as established in their lab . As for TEM visualisation - I prefer non-purified plant extracts since the purification procedures are biased against very short rods and disks which don't sediment as well. On the other hand several precipitation, peleting and resuspension steps can break full-sized rods into shorter ones.
I dont have experience with neither of SBMV ,BPMV and CP-TMV, however I have worked with various mutants of regular TMV which is probably quite similar to CP-TMV. Even though TMV can alsobe purified using chlorofom clarification, the yields tend to be somewhat lower than with PEG precipitation (Gooding & Hebert, Phytopathology 57: 1285, 1967) at least in my hands. Also chlorofom is not a chemical people want to work with in litre volumes nowadays when everybody uses kits and most of the work is done sitting next to a PC :) . However the chlorofom clarification was a method of choice for mutants that do not form ful-sized particles but stacked disks.
As for the chlorofom addition at the end of purification - I dont think I can find a reference. I did that couple of times when the virus pellet was particularly green with minute amounts of chloroform eg. 100 µl per 1 ml of virus prep. It probably has some yield penalty. However since most of the green contaminants are quite insoluble, they can also be removed by 10 min centrifucation in eppendorf tube from aqueous solution. Hope that I have answered at least partially your enquiry,
best regards Tomas
Dear Tomas, I thank you for this (these) explanation(s). Maybe Tommy Zhou can remember (or find out) the reference for the "chloroform as a last step in purification of TMV" mentioning paper....I have seen your marvellous TMV-TEM images but never had need or possibility to purify any virus preps so far the way it is done /has been shown by you. Great work....Thank you so much, best regards and wishes for the future work, Wolfgang
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