Hi everyone!
I have been working on a protein with mol. wt. of 45 kDa. This protein is present in inclusion bodies, which I have refolded and purified. I wish to carry out cryo EM experiments regarding which I have following questions:
1) Is it possible to carry out Cryo EM experiment with protein of 45 kDa size?
2) Is it necessary to carry out negative staining EM before going for Cryo EM experiment?
3) What is the concentration of protein that is required for cryo EM experiments?
4) I have attached the SDS PAGE image of refolded and purified protein. Is the protein pure enough for cryo EM experiment?
Thank you
Hi,
As Sebastian Schmitt, I would also say that the protein you're working with is too small to get a structure using CryoEM, unless it forms oligomers. You can check that with gel filtration, native PAGE or any other suitable method. If you want to get the structure, I would say that around 200kDa is currently usually considered as the minimal size for CryoEM. There are a few smaller proteins for which it worked... but only a few... and I'm not sure the "particules" that were resolved with the EM had realy only that size.
But maybe you want to use your protein in other experiments you can do with cryo EM (i.e. in context of larger complexes)?
The smallest protein structure reported till now is Haemoglobin 64kDa . The problem of small proteins have been partly curtailed by use of Volta phase plates. But yes anything less than 200kDa still remains challenging!
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