[3dem] protein complex crosslinking for EM: GraFix

Harris, Audray (NIH/NCI) [E] harrisau at mail.nih.gov
Tue Jul 1 06:08:58 PDT 2008


Dear Alexandra:
Kastner et al. (Nat Methods, 2008 Jan:5(1):53-5  have worked on this issue
by using cross-linking coupled with gradient centrifugation to stabilize
complexes for cryo-electron microscopy. The method is termed "GraFix".
I hope this reference helps you.

Audray Harris
Research Fellow
Laboratory of Cell Biology
Biophysics Section and Unit
National Cancer Institute, NIH


-----Original Message-----
From: 3dem-bounces at ncmir.ucsd.edu on behalf of Alexandra Deaconescu
Sent: Tue 7/1/2008 1:49 AM
To: 3dem at ucsd.edu
Subject: [3dem] protein complex crosslinking for EM
 
Dear EM enthusiasts:

I am working on a protein complex that appears to dissociate upon  
dilution for EM grid preparation. I was therefore thinking of using  
some sort of heterobifunctional crosslinker (something other than  
glutaraldehyde that would probably generate a big mess) to stabilize  
the complex and then purify it by gel filtration. I was wondering if  
anyone has and would like to share his/her experiences, or point me  
to some good papers where this has been done before...

I would greatly appreciate your help.

Best,
Alexandra






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