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

[Reinhard Rachel]

>>> 30.03.2020 at 05:15:
> 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.

Dear Satoru, 
You do not state the source of your protein-RNA complex - but most likely,
under physiological conditions, it is an 'intracellular' complex. Which cell -
prokaryotic i.e. bacterial, or archaeal? or eukaryotic? organelles of a
eukaryote? - this already might be important to know. Which temperature is
optimal? important to know, too. 
Anyway. My argument is - your buffer. 
IF this complex is located inside the cytoplasm of any given cell, the optimal
pH would be close to pH 7.0 (with quite some variations, depending on the
physiology of the cell!). This means that the conc of H+ ions ideally might be
10 times higher than in the buffer you are using. - second: NaCl as main salt
used in your buffer (150 mM) is close to what is optimal to EXTRAcellular
environments but NOT to intracellular conditions. I doubt that this is ideal
for your complex under investigation. Inside cells, you have a balance of K+
and Na+ ions, with 3 to 10(0) x higher conc of K+ (!!). In addition, there is
some Cl- found inside cells, but the dominant counterions are Phosphate groups
from nucleic acids and metabolites, and carboxylates (from metabolites).
Whether Cl- can be used? They may - but if you fail, you have to think about
this. 

thus, I would use HEPES at pH 7; use the optimal temp for your complex before
starting the freezing process (which organism at which 'optimal' temp? this
influences the real pH); and a balanced amount of lots of K+, little Na+, and
counterions (you may start with Cl- salts, simply for ease of use; but later,
you may think about phosphates and carbonates).  
and adding EDTA and DTT is common use, but whether this physiological? it
might help. 
EDTA substitutes for the fact that inside a cell, there are many metabolites
with similar 'complexing' functions; DTT substitutes for the fact that inside a
cell, there are usually quite complex systems for keeping the Redox state in
balance (i.e. reduced). This again depends on your type of cell. 

just my two cents ... 
kind regards - Reinhard
 

-- 
Prof. Dr. Reinhard Rachel
University of Regensburg
Centre for EM / Anatomy
Faculty of Biology & Preclin. Med.
Universitaetsstrasse 31
D-93053 Regensburg - Germany
tel +49 941 943 -2837, -1720
mail reinhard.rachel at biologie.uni-regensburg.de
office: VKL 3.1.29 
member of the IFSM board

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