[3dem] how to do cryo-EM sample preparation with the sample
heated to 60 degreeL JSB paper
harrisau at mail.nih.gov
Tue May 15 06:08:35 PDT 2012
I think that some cry-EM labs have done this type of thing with some
interesting results. Try the reference below. It may be helpful
Audray Harris Ph.D.
A Thermally Induced Phase Transition in a Viral Capsid
Transforms the Hexamers, Leaving the Pentamers Unchanged
James F. Conway1, Naiqian Cheng2, Philip D. Ross3, Roger W. Hendrix4,
Robert L. Duda4,
and Alasdair C. Steven2,
J Struct Biol. 2007 May; 158(2): 224–232
On May 14, 2012, at 8:49 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 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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