We are studying a protein that forms tubules in vitro. We have some interesting results from negative staining electron microscopy performed on purified recombinant protein. Can someone please point me to a publication that illustrates common artefacts seen in negative staining EM?
Dear Ho Leung Ng,
I am not really aware of (an) article(s) dealing explicitely with examples of artificial altered formation of (pritein)structures due to negative staining.
As an example I've in my files only (for now):
Lei ZHANG et al, 2010: , dealing also with artifacts (cf. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2853450/ ).
That negative staining naturally will induce artificial structures you certainly have found in the protocol http://mitchison.med.harvard.edu/protocols/microtubules/Negative%20Stain%20Electron%20Microscopy%20of%20Microtubules.pdf too.
Also see: Negative Staining, by Baschong and Uebi, Cell Biol, 2006: http://digitus.itk.ppke.hu/~cseri/edu/Books/Cell%20Biology/Volume%203/Part%20B/Chapter23.pdf
Apologize, if nothing at all I can be of help here....[unless you show at least one image of your structures (:-)) ]
Best of luck, regards, Wolfgang
Dear Ho Leung Ng,
I am not really aware of (an) article(s) dealing explicitely with examples of artificial altered formation of (pritein)structures due to negative staining.
As an example I've in my files only (for now):
Lei ZHANG et al, 2010: , dealing also with artifacts (cf. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2853450/ ).
That negative staining naturally will induce artificial structures you certainly have found in the protocol http://mitchison.med.harvard.edu/protocols/microtubules/Negative%20Stain%20Electron%20Microscopy%20of%20Microtubules.pdf too.
Also see: Negative Staining, by Baschong and Uebi, Cell Biol, 2006: http://digitus.itk.ppke.hu/~cseri/edu/Books/Cell%20Biology/Volume%203/Part%20B/Chapter23.pdf
Apologize, if nothing at all I can be of help here....[unless you show at least one image of your structures (:-)) ]
Best of luck, regards, Wolfgang
For negative staining, two major problems are (1) Collapse structure (2) Preferred orientation. For negative staining, Ohi 2004 http://www.ncbi.nlm.nih.gov/pmc/articles/PMC389902/ But most of the cases, good class averages give very good information (most of the cases we can trust those results) but negative staining is not good for 3D reconstruction.
Negative staining is a great method for an initial sample screening and gives a distinct flavour of how your protein looks and behaves, but you have to exercise extreme caution in interpreting these types of data. Ultimately cryo is always better because you don't get the same range of dehydration, shrinkage, sample skewing, partial staining, incomplete penetration and preferential orientation you always get to some degree with negative stain.
There's no large collection of unusual negative stain oddities that I know of (maybe there should be), but when dealing specifically with fibres there are a few things you should consider:
- Stain streaking. In several publications with TEM as a minor component people have sometimes attributed stain crystals as fibres or tubules. Be careful here.
- A mixture of negative and positive staining. Unusual surface properties can make a fibre exhibit negative and positive staining at the same time. Impossible to make sense of, but look great.
- Shallow stain penetration. Lots of fibre preps need high salt concentrations to keep them soluble and this can cause problems with stain.
- Is your fibre hollow? If so, your negative stained images will be flattened and any diamter measures you infer will be wider.
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