I am planing for cryo-em of a AAA+ ATPase (natively hexamer of 360 Kda)
properties> forms multimer of hexamer at concentration above 2mg/ml and below Nacl 500mM.
Also show denaturation on freezing(required to freez for transporting to cryo-EM facility)
Earlier I did SAXS of this protein and got Aggregated profile from low to high concentration. buffering condition was 150Mm Nacl, 5% Glycerol ,10mM Arginine, 4mM bME, 25 mM HEPES 7.5pH.
i want to know Whether this buffering condition suits cryo-EM requirement?
If SAXS is showing Aggregated profile, is it possible get single hexamer images from cryo-EM in same buffering condition(given above) ?
what are the precaution required to take care during sample prepration?
this will be my first time on cryo-EM.
All the assistance and suggestion are welcome
Regards
Ketul Saharan.
Glycerol can cause problems for electron microscopy, but 5% may still be ok. If you see that your sample starts to look strange with bubles this is likely due to the glycerol, which is sensitive to radiation. An idea could be to prepare at least some sample that does not contain it or to be prepared to dialyse some sample before freezing.
Spinning down the sample may also remove some of the aggregates. A good way to optimize your sample would be to start with negative stain. This is much faster so you can try a few things before doing cryo. It will already show if the aggregates are a problem, or whether you have still enough single protein to proceed. It will also alow you to optimize the concentration of protein.
Sample preparation for cryo-EM is mostly trial and error, so the best advice is basically just to have a look and then go from there.
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