[3dem] how to do cryo-EM sample preparation with the sample heated
to 60 degree
yanzhang
yanzhang at moon.ibp.ac.cn
Mon May 14 17:49:49 PDT 2012
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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