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

[linjunn at gmail.com]

Or

=B7=A2=D7=D4=CE=D2=B5=C4 iPhonee

=D4=DA 2012-5-16=A3=AC1:1hgty7=A3=ACShaoxia <shaoxia at mrc-lmb.cam.ac.uk> =
=D0=B4=B5=BD=A3=BA

> Hi Yan Zhang,
> Tt
> Have you rrr temperature firstly? If it works at room temperature, =20
> the problem may just be the evaporation rate which is much high at =20
> 60 degree.
>
> Shaoxia Chen
> MRC-LMB
>
> On 2012-5-15 17:16, David Belnap wrote:
>> Yanzhang,
>>
>> Did you use 100% relative humidity, or get as close as you can to =20
>> 100%?  That may be important for avoiding rapid evaporation at =20
>> 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 =20
>>> sample preparation: During the sample preparation process using =20
>>> FEI vitroboot system, I also needed to  heat the sample to 60 =20
>>> degree inside the cavity of vitroboot, because I wanted to see if =20=

>>> the structure of my protein would be changed after heated to 60 =20
>>> degree. But when I observed the heated cryo-em sample using =20
>>> electron microscopy, I found a strange phenomenon: 1. there was no =20=

>>> ice in most of the holes; 2. Only very very few holes located at =20
>>> the boundary of some squares of the grid have ice; 3. For the very =20=

>>> few holes with ice, there were very little samples located at the =20=

>>> inside boundary of the holes.
>>>
>>>            I tried many different conditions of blotting time and =20=

>>> blotting force, but no chang for the sample.
>>>
>>>            I'm not sure why I can't get a good heated cryo-em =20
>>> sample. Then I tried to heated the sample in the water-bath first, =20=

>>> then add the sample to the tweezers in vitroboot (vitroboot also =20
>>> heated to 60 degree), but nothing changed, I still can't get a =20
>>> good sample to collect data.
>>>
>>>           p.s. I used the quantiful film for cryo-em sample =20
>>> preparation. I once tried the carbon film for this heated cryo-em =20=

>>> sample preparation, but the results even worse: the films were =20
>>> totally broken.
>>>
>>>           Even I repeat the experiment many times, but I got =20
>>> nothing. So please anyone who have the experiences of doing heated =20=

>>> 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
>>>
>>>
>>>
>>>
>>>
>>>
>>> _______________________________________________
>>> 3dem mailing list
>>> 3dem at ncmir.ucsd.edu
>>> https://mail.ncmir.ucsd.edu/mailman/listinfo/3dem
>> =3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=
=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D
>>  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
>> =3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=
=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D
>>
>> _______________________________________________
>> 3dem mailing list
>> 3dem at ncmir.ucsd.edu
>> https://mail.ncmir.ucsd.edu/mailman/listinfo/3dem
> _______________________________________________
> 3dem mailing list
> 3dem at ncmir.ucsd.edu
> https://mail.ncmir.ucsd.edu/mailman/listinfo/3dem