Questions about minipurification
What can I do if I don't have an ultracentrifuge?
Be assertive and get a grant or be resourceful and borrow one. If you develop a simpler technique that concentrates virus more selectively I'd like to be the first to know
Shouldn't one extract under physiological conditions?
Viruses have to come apart (uncoat) under physiological conditions.
Why does my final prep contain rubisco?
Rubisco is the most abundant soluble protein. It's large (20S) and pellets in the ultracentrifuge. Ultracentrifugation concentrates viruses relative to rubisco in the ratio of sedimentation coefficients.
Is this technique better than dsRNA analysis?
In general yes. Viruses make vastly more capsid protein than dsRNA and do so more reproducibly. Keep in mind that minipurified preps contain host proteins which require interpretation. Minipurification is more versatile than dsRNA analysis.
Why are my pellets so large?
Perhaps you've got TMV, but more likely your plant has gums. These are overlooked in virus purification literature because reviewers would say "prove it" which, of course, you couldn't (at least to their satisfaction).
Shouldn't samples be kept cool (close to 0oC)?
Viruses are stable below 45oC. In no case (unless thiols are present) do capsids proteolyze. Higher temperatures reduce solution viscosity (and centrifugation time; see viscosity vs. temperature tables for water).
Are citrates really that good for extracting viruses?
Apparently yes. Citrate likely chelates Ca. Citrates are highly stabilizing (Hofmeister series). Conversely they're good precipitants; so we're lucky that viruses are soluble. Citrates may precipitate contaminants.
I have a buffer and pH that work better for my virus
By all means use it. Minipurification conditions are meant for the widest range of viruses. For a specific host and specific virus one would be foolish not to optimize conditions.
Why not use a smaller sample and smaller volumes?
If you can find a convenient grinder and if you can recover virus pellets, it's worth considering. Small tissue samples increase "sampling error" (i.e. inadvertently selecting healthy tissue).
How reliable is minipurification for diagnosis?
If you diagnose moderate to high concentration viruses in "ideal tissue", it's highly reliable. You can make it (and other methods) totally unreliable by diagnosing low concentration viruses in old and/or abused tissue.