[3dem] 回复: protein can't go to holes of cryo EM grids

[SSS Sasha]

Hi Chris,

Thanks for your reply!

For lacey carbon, I atteched the atlas and FFT iamges here (ignore the yellow box). There are big and small holes. I did try several grids with different blocking time. I felt no matter thick or thin ice, they behave quite similar. If it is too thin, there is no particles any more. Let me know how do you think my FFT image.

Yes, I would appreciate if you can send me some protocols and reagent list.😀

Thanks
Shasha
________________________________
发件人: Christopher Arthur <cparthurphd at gmail.com>
发送时间: 2022年10月10日 14:48
收件人: SSS Sasha <sss_jiandan at outlook.com>
主题: Re: [3dem] protein can't go to holes of cryo EM grids

Hi Shasha,
Looking at your images, it looks like you have protein on the lacey grids, you may just have too thick of ice. Can you look at thinner areas on the lacey grids? If you take an FFT of the image, do you see the light colored ice ring? It should be in the 3.5A range. This is usually an indicator that your ice is a bit too thick. You can also increase your defocus just to verify that you have protein present.
For trying to get your samples into holes. What has worked for me on occasion is to use Au foil grids, and try a self-assembled monolayer of lipoamide. This binds to the Au surface and helps to inhibit the sample from binding to the substrate.
I am happy to send you some protocols and reagent list if you are interested.

Best,
Chris



Christopher P. Arthur, Ph.D.
(c) 858-883-8303


On Mon, Oct 10, 2022 at 12:47 PM SSS Sasha <sss_jiandan at outlook.com<mailto:sss_jiandan at outlook.com>> wrote:
Dear all,

I was trying to freeze cryo EM grids for a protein ~ 0.73MDa under several conditions, but it didn't work well. I make a brief summary here. Does anyone have any suggestions? Thanks in advance!

Holly grids CF-1.2/1.3-4Cu-50: plasma cleaning for 10s using O2,Ar2            Protein can't go in to the hole

Lacey carbon 400mesh Cu:  plasma cleaning for 15s using O2, Ar2        Contrast is quite low, hardly see the particles

GO on Quantifoil: plasma cleaning for 15s using H2, O2              Protein can't go into hole

Best regards,
Shasha
[cid:183c383c8cdd2a6827e1]
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