[3dem] RE: EM sample in high salt

[Henning Stahlberg]

Hi,

That cryo-neg. staining method was developed by Marc Adrian et al., in  
the Dubochet lab at Lausanne.
The first publication was
Adrian, M., Dubochet, J., Fuller, S.D. and Harris, J.R. (1998) Cryo- 
negative staining. Micron, 29, 145-160.
As far as I know, the earliest application of that method to study a  
new protein appeared in PNAS 96, 6787-6790 (1999),
and Sacha de Carlo nicely analyzed the possible resolution when  
applied to GroEL, here:
De Carlo, S., El-Bez, C., Alvarez-Rua, C., Borge, J. and Dubochet, J.  
(2002) Cryo-negative staining reduces electron-beam sensitivity of  
vitrified biological particles. J. Struct. Biol., 138, 216.

Henning.


On Jul 9, 2008, at 10:08 AM, Peijun Zhang wrote:

> 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
>