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

[yanzhang]

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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