[3dem] DNA-protein cross-linker

[Zannati Zaoti]

Hello Daniel,

Thank you for your response.
I don’t know much about it if UV-induced crosslinkers may cause any damaging effects on the sample.


Regards
Zaoti

From: Daniel Asarnow <asarnow at msg.ucsf.edu>
Date: Wednesday, 24 January 2024 at 12:21 pm
To: Zannati Zaoti <z.zaoti at uq.edu.au>
Cc: 3dem at ncmir.ucsd.edu <3dem at ncmir.ucsd.edu>, 3dem-request at ncmir.ucsd.edu <3dem-request at ncmir.ucsd.edu>
Subject: Re: [3dem] DNA-protein cross-linker
Perhaps ultraviolet light induced crosslinking would be worth a shot.

Best,
-da

On Tue, Jan 23, 2024 at 6:08 PM Zannati Zaoti <z.zaoti at uq.edu.au<mailto:z.zaoti at uq.edu.au>> wrote:
Dear all, I am looking for advice from experienced individuals who have performed cross-linking of DNA-bound protein complexes for cryo-EM sample preparation. My work involves a protein that can bind to dsDNA and form filaments; however, these
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Dear all,

I am looking for advice from experienced individuals who have performed cross-linking of DNA-bound protein complexes for cryo-EM sample preparation.
My work involves a protein that can bind to dsDNA and form filaments; however, these filaments are flexible and tend to fall apart during grid preparation. As a result, I cannot find any filaments during the cryoEM screening.
Thereafter, I have tried using glutaraldehyde and formaldehyde as cross-linkers to stabilize the filaments, but they have made the filaments even more unstable. At present, I am experimenting with DMS, but it seems that DMS neither makes the filament unstable nor stable enough for cryo-conditions.
Any advice or comment would be highly appreciated. Thank you!


Regards
Zaoti


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