[3dem] RE: EM sample in high salt
Henning Stahlberg
HStahlberg at ucdavis.edu
Wed Jul 9 10:18:59 PDT 2008
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
>
More information about the 3dem
mailing list