[3dem] How to improve particle distribution on GO-coated ultrAufoil

[Satoru Machida]

Dear all

I prepared cryo grids for single particle analysis for 600 kD protein-RNA complex. The particles are overcrowded. After dilution, the particles are not separated. When I diluted it further, holes became empty. Gel filtration makes a single peak, but on the grid they stick together. I already tried glycerol, DMSO, amphipols, salt concentration 70-400 mM. None of them worked well. The buffer contains 150 mM NaCl, 10 mM HEPES pH8, 0.5 mM EDTA, 4 mM DTT. The sample had about 1mg/ml, blotted for 3 sec , force 0, 100% humidity, 22C on Ultraufoil R0.6/1.0 coated with graphene-oxide. The image was taken by Tecnai12. The particles are hard to get into the holes without graphene-oxide.
Any expert suggestions will be helpful.

Kind regards
Machida

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