[3dem] DNA-protein cross-linker

[Alexandre Cassago]

Dear Zaoti, 

A long time ago we used DSS crosslink (attached) and it worked very well. The protein complexes, depending on their isoforms, assembled long filaments with increased activities compared to the well-known tetramers, however when we reduced the concentration for NS microscopy essay the filaments disassembled. Crosslinking helped us visualize naturally occurring filaments, but at lower concentrations required by NS microscopy.

Hope it helps you.


Best, 
Alexandre 




> On Jan 23, 2024, at 18:08, Zannati Zaoti <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 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     
>  
>  
> _______________________________________________
> 3dem mailing list
> 3dem at ncmir.ucsd.edu <mailto:3dem at ncmir.ucsd.edu>
> https://mail.ncmir.ucsd.edu/mailman/listinfo/3dem

-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://mail.ncmir.ucsd.edu/pipermail/3dem/attachments/20240124/b7631080/attachment-0003.html>
-------------- next part --------------
A non-text attachment was scrubbed...
Name: DSS-crosslink.pdf
Type: application/pdf
Size: 174869 bytes
Desc: not available
URL: <http://mail.ncmir.ucsd.edu/pipermail/3dem/attachments/20240124/b7631080/attachment-0002.pdf>
-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://mail.ncmir.ucsd.edu/pipermail/3dem/attachments/20240124/b7631080/attachment-0004.html>
-------------- next part --------------
A non-text attachment was scrubbed...
Name: Ferreira_2013.pdf
Type: application/pdf
Size: 3030971 bytes
Desc: not available
URL: <http://mail.ncmir.ucsd.edu/pipermail/3dem/attachments/20240124/b7631080/attachment-0003.pdf>
-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://mail.ncmir.ucsd.edu/pipermail/3dem/attachments/20240124/b7631080/attachment-0005.html>