[3dem] RE: EM sample in high salt

[Peijun Zhang]

Thanks to all that replied. You might find this useful.

 

The question was: How to make good cryoEM specimen for the sample only
stable in high salt (1M Nacl)?  The following is a summary of the replies.

 

1)       use GraFix method is: Kastner B et al. GraFix: sample preparation
for single-particle electron cryomicroscopy. Nat Methods. 2008
Jan;5(1):53-5. Epub 2007 Dec 23.  

2)       cross-linking with low glutaraldehyde concentrations.

3)       freeze the sample in high salt. Nature (2001), vol 409 1047  (note,
S/N was actually poor).

4)       using the cryo-negative staining protocol at (~0.84M Ammonium
Molybdate) developed by Sacha De Carlo et al.

5)       dilution on a holey grid:  put your sample onto one side of a holey
grid and put a large droplet of buffer at the desired salt concentration (or
even a bit lower) on the opposite side of the grid. Then blot from the back
side (the side with the large buffer droplet)

Best,

 

Peijun

 

  _____  

From: Peijun Zhang [mailto:pez7+ at pitt.edu] 
Sent: Monday, July 07, 2008 6:16 PM
To: '3dem at ncmir.ucsd.edu'
Subject: EM sample in high salt

 

Hello all,

 

I have an EM specimen which only stabilizes in high salt buffer (1M NaCl).
I would like to carry out cryoEM imaging at lower salt conditions.  Does
anyone have a good protocol to reduce salt to 100mM or less without
compromising the specimen?  

 

Thanks so much,

 

Best,

 

Peijun

 

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