[3dem] how to do cryo-EM sample preparation with the sample heated to 60 degree

[David Belnap]

Yanzhang,

Did you use 100% relative humidity, or get as close as you can to 100%?  That may be important for avoiding rapid evaporation at higher temperatures.

David


On May 14, 2012, at 6:49 PM, yanzhang wrote:

> Dear all,
>            I encountered a problem when I was doing the cryo-EM sample preparation: During the sample preparation process using FEI vitroboot system, I also needed to  heat the sample to 60 degree inside the cavity of vitroboot, because I wanted to see if the structure of my protein would be changed after heated to 60 degree. But when I observed the heated cryo-em sample using electron microscopy, I found a strange phenomenon: 1. there was no ice in most of the holes; 2. Only very very few holes located at the boundary of some squares of the grid have ice; 3. For the very few holes with ice, there were very little samples located at the inside boundary of the holes.    
>         
>            I tried many different conditions of blotting time and blotting force, but no chang for the sample.    
>        
>            I'm not sure why I can't get a good heated cryo-em sample. Then I tried to heated the sample in the water-bath first, then add the sample to the tweezers in vitroboot (vitroboot also heated to 60 degree), but nothing changed, I still can't get a good sample to collect data.
>         
>           p.s. I used the quantiful film for cryo-em sample preparation. I once tried the carbon film for this heated cryo-em sample preparation, but the results even worse: the films were totally broken.        
>           
>           Even I repeat the experiment many times, but I got nothing. So please anyone who have the experiences of doing heated cryo-em sample preparation, please give me suggestions!
>         
>          Thank you! 
> yanzhang
> 
> yanzhang at moon.ibp.ac.cn
> 
> Institute of Biophysics,
> Chinese Academy of Sciences,
> 
> 15 Datun Road,
> Chaoyang District,
> Beijing, China
> 
> Tel(o): 64888137
> 
> 
> 
> 
> 
> 
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============================================
 David M. Belnap
 Department of Chemistry and Biochemistry
 Brigham Young University
 Provo, Utah 84602  USA

 Phone:  801-422-9163
 FAX:      801-422-0153
 David.Belnap at byu.edu
============================================