I am trying to do cryo-EM of a protein but when i see the images i see them aggregated. I do not think my protein is aggregated since the same sample of protein crystalized well and in gel filtration it shows a nice peak corresponding to the expected molecular weight. what could be the posible problem.
Maybe your sample is getting dehydrated during the freezing process? Do you use a Vitrobot or similar device with humidity control?
Also, if you are loosing solvent to evaporation, the concentration of any co-solutes (e.g. salts, buffer, etc.) will increase and may become unfavourable for the protein to remain monomeric / non-aggregated.
Hi Anish Thachangattuthodi , Sample preparation by the commercially available vitrobot is not as trivial as we generally think. Freeezing samples leads to molecular motions which lead to protein preferentially moving towards air-water interface. This problem manifests in the form of denaturation of the protein/ or classical preferred orientation on the grid.
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