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

[Audray Harris]

Dear Yanzhang:
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.
NCI
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
>
> yanzhang at moon.ibp.ac.cn
>
> Institute of Biophysics,
> Chinese Academy of Sciences,
>
> 15 Datun Road,
> Chaoyang District,
> Beijing, China
>
> Tel(o): 64888137
>
>
>
>
>
>
> <ATT00001.txt>

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