[3dem] protein complex crosslinking for EM: GraFix

[Daniel Levy]

Dear Alexandra
You may also try to bind your protein to a functionalyzed lipid layer 
containing a lipid ligand, e;g. NiNTA-DOGS for an Hist protein, that would 
protect the complex at the air/water interface
the procedure is well described in the paper of Kelly, Dukovsky and Walz, 
2008. PNAS, 4703
daniel


At 15:08 01/07/2008, Harris, Audray (NIH/NCI) [E] wrote:
>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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>
>
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Dr. Daniel Lévy
"Structure of membrane proteins by electron microscopy"
Institut Curie
UMR CNRS 168
11 rue P.M.Curie
75005 Paris, France
please note the new phone number
Phone:00 33 1 56 24 67 82
Fax:00 33 1 40 51 06 36
Email:daniel.levy at curie.fr