[3dem] specimen preparation

[Xinghong Dai]

Hi Kimberley.

I had similar problems before with NP40-containing samples. The reason was
because the detergent dramatically decreased the surface tension of the
solution, so it is much easier to blot away than regular PBS-only samples.
The lipid in your sample might have the same effect. 

For you 2nd sample, you may consider about the thickness of the ice. I don¡¯
t know the size of your particles, but if they are bigger than the thickness
of the ice, they cannot stay in the ice due to size-exclusion effect. 

I would suggest you try manual freezing instead of vitrobot. In vitrobot,
the blotting occurs only at one edge of the grid, so it is hard to get even
ice with short blot time.  With manual blotting, you can make your filter
paper parallel to the surface of the grid to get even thickness of ice.
Anyway, that¡¯s how I solved my problem. 

Good luck! 

  

 

Xinghong Dai @ UCLA



·¢¼þÈË: 3dem-bounces at ncmir.ucsd.edu [mailto:3dem-bounces at ncmir.ucsd.edu] ´ú
±í Kimberley Cheng
·¢ËÍʱ¼ä: Wednesday, November 06, 2013 5:35 AM
ÊÕ¼þÈË: 3dem at ncmir.ucsd.edu
Ö÷Ìâ: [3dem] specimen preparation

 

Dear all,

I have difficulties preparing cryoEM specimens of my samples, and I would
appreciate any help I can get. Both samples contain lipid nanoparticles, and
I use a Vitrobot to prepare the frozen-hydrated specimens. The problems are
the following

Sample 1: Majority of the grid has no ice at all. If I am lucky, a small
region of the grid (1/4 of a grid square) is covered with ice that is very
thick. What can be the problem? Any advice on how to improve the procedure?

Sample 2: Ice thickness and particle distribution is good. The concentration
is, however, very low. Any suggestions on how to increase the on-grid
particle concentration? I have already tried preparing the sample on
continuous carbon instead of perforated carbon film.

I am very interested in hearing your opinions in this. 

Thanks in advance,
Kimberley Cheng




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