We collected some wt TMV virions from tobacco plant leaves. We used 2X PEG precipitation and got a very large amount of virions on SDS-PAGE. When we proceeded to TEM to visualize the rod-shaped virions, I noticed that they were of different lengths. They varied from 70nm - 500+nm, mostly around 400nm. We tried a few different samples and found that this was very common. Does anyone know if they are supposed to vary in size? Are there any steps during PEG precipitation that can cause virions to break into small pieces?
Hi Tommy, from the literature is known that the most common length is about 300 nm but that there are always shorter and longer particles are also found. Cheers, Nadine
Hi Tommy. I do TEM of TMV particles almost on daily basis. Yup, they can vary in length, depending on the way you process the samples. The length of intact particle containing TMV RNA is really 300 nm, however you usually get a mixture of shorter/longer particles as well. Things that can have impact - extraction of the leaves- bead cell lyser vs gently crushing the leaves with mortar and pestle (shorter/longer), ionic strength and pH of buffer used for extraction. Another step is the PEG precipitation followed by pellet resolubilization. Some of the particles break into smaller pieces during this step. Do you immunocapture your virus or just attach the particlesdirectly onto the grid? I have found that when doing immunocapture I get higher proportion of shorter particles, suggesting that without the antibody the longer particles stick better to the grid, while the shorter get washed away. The stain and pH used for TEM can affect the length/stability. Also the calibration of your microscope can effect that - I have noticed that the same particle observed at different magnification can vary slightly in length and this difference is systematic, ie I know that the 56kx magnification gives me about 3% longer measurement than 71kx. YMMV
Besides breaking, the particles have a tendency to attach to each other head-to-tail , thus making particles longer than 300 nm possible. You can measure randomly several hundred of them and then calculate histogram or other statistical distribution. If you really want the 300 nm fraction you will have to do purification using gradient centrifugation and/or gel filtration and avoid all steps where the virus will end up in the pellet.
Just adding a picture from yeasterday - purified virions containing sligthly longer RNA (about 7%).
56kx, ammonium molybdate+trehalose, pH7,0 captured on fromvar coated grid with agdia polyclonal antibody, this has also been precipitated with PEG, if you do it directly from leaf sap, you end up with plenty of 1-2 layer discs facing you and looking like doughnuts :)
Normal length of TMV is established by testing the lenghts of dip preparations rather than PEG precipated ones. When this is done is there is a ceratin level of varition between particles and the size of 300 is the most frequent one.
So go ahead and check first negatively stained particles d from a dip preparations, if you see a clear band higher than the normal size 300 nm ones, you may have an assembly mutant, but I doubt it, its probably your purification proecure.,
negatively stained leaf dip of TMV will usually appear as 300 nm rigid rods. but breakage, can happen due to processing like ultra centrifugation etc
Hi Tommy,
Certainly! the length of TMV can vary but most common length is about 300 nm. Your entire procedure and precaution may have great impact on ideal length. Sometimes breakage can happen and cause the particle get shorter and in contrast attachment of neighboring particles end to end may make the particle longer than usual. So don't worry about that! If you want to get 300 nm please follow the standard purification procedure very carefully.
Tommy, if you want to know the actual length of your TMV isolate the best way is to use unprocessed, or minimally processed samples. For example, using immunocapture floating an Ab coated TEM grid in a drop of buffer winth some chopped fragments of infected leaf blade. Then wash well and stain. Good lack!
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Cell Biology
- Culture Cells
- Cell Line