I am trying to get a good images of my protein and see by TEM. Sometimes I get a good images however others not. After many rounds, I conclude that the problems fall into the UAc staining, summarizing 1) I get a background of UAc crystals which form a network where I cannot see clearly the difference between protein and crystals. 2) the UAc acts as blanket-like covering my protein avoiding see anything.
I always do the same; final solution of my protein--> super pure H2O. Freshly prepare 2% of AcU solution in water, filter 0.22um,
Are there some tricks or explanation for that?
UA precipitation in negative stains can be caused by residual buffer salts from the specimen (especially phosphates), excess UA stain fluid left on the film surface before drying, and by staining with high concentrations of UA. Try dialyzing the sample to remove the buffer or substitute a different buffer type, use UA solutions of 0.1% to negative stain, and carefully remove as much stain as possible from the grid before drying.
Thank you very much, I really apreciate your answer.
finally I have concluded that the problem was, as you said, the residual phosphate, I have tested with other sample that I know is free for phosphate and i had not seen any precipitate.
Álex
Phophate causing precipitation of metal cations is alwaays a problem. Here is the technique used by the US CDC, similar to the way I did it: http://emergency.cdc.gov/agent/smallpox/lab-testing/pdf/em-rash-protocol.pdf
When you need to draw off the fluid, make long narrow wedges of highly absorbent fine filter paper-- don't worry about sucking off all your sample, plenty will have stuck to the coated grid. You can also centrifuge the UrAc with a tabletop centrifgue or a microfuge for several minutes before use, especially if you disturbed the bottle before use.
Prior to staining, it is important to ensure that the buffer in which the sample is suspended is not salt rich (e.g. phosphate buffer-saline (PBS)), as simultaneous deposition of the salts and the sample might otherwise be experienced, ultimately resulting in unwanted diffraction events (salts are crystalline, thus they are characterized by very high contrast, which disturbs the weaker contrast of bio-specimens; they may also mix with the staining solution). For this reason, distilled water or diluted 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) are normally the best choices.
reference:
Our lab uses 1% alcoholic uranyl acetate, wash well in 3 -50 ml of beakers of boiled de-ionized water, 40 dips each. With 4th beaker to rinse tweezers in between staining. The syringe filter we use is - 0.1 micron, 13 mm in diameter , and 1.0 ml syringe.
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