[3dem] Re: 3dem Digest, Vol 45, Issue 16
DocDave50 at aol.com
DocDave50 at aol.com
Sat May 28 08:49:03 PDT 2011
Hi Kimberley,
It is interesting that folks have identified Trehalose as a sugar that
appears to preserve proteins. If I'm not mistaken, this property was
discovered by Gordon Jendrasiak (sp?) and his wife back in the 70's or possibly
earlier. You would think that a sugar is a sugar...some carbon, a few
hydroxyls here and there and some kind of "chair like" configuration...they found
that the physical chemical properties of Trehalose replaces hydrogen
bonding requirements of all kinds of biological materials (lipids and proteins)
much better than any other sugar. In one experiment, they prepared a lipid
dispersion in Trehalose versus other sugars and found that when dried
completely, the Trehalose samples had identical physical chemical behaviours
(e.g., melting transitions) observed with the fully hydrated samples. Some
other folks from NIH...can't remember the authors name off hand...tried this
with proteins and found that protein structure upon dehydration remained
constant.
As for sucrose concentration, we had trouble blotting sucrose when the
concentration exceeded 1-2% due to the viscosity of the solution. Hence, the
ice tended to be much thicker than with buffer alone. 1000 A ice was almos
t impossible to achieve with this level of sucrose. One of the comments
suggested replacement with Trehalose. I've established a method for Cryo-EM
based on the "Spin Column" approach sample desalting. In this method, 50
ul G-50 columns are prepared in modified in P200 pipette tips (very small
porous disc pressed into the base of the pipette tip and the tip end cut at
the base of the 'frit'). Using a P200 pipetter, the void volume is gently
expressed out of the G-50. Your sample volume to be added to your glow
discharged grid for Cryo-EM (4-6 ul) is added to the middle of this tiny column
and, using the P200 pipetter, you sample is expressed through this micro
column right onto the grid. Buffer exchange occurs over a second or two and
your sample can be freeze quenched within the time it takes to generally
blot the sample prior to freeze quenching. At present, I've not done
anything to publish this approach but it works perfectly for buffer exchange and
your sample is freeze quenched in liquid ethane within seconds of exchange.
If you'd like, I can provide a more thoroughly detailed description.
This will remove any issues you might have with samples in high sugar
concentrations limiting contrast in your Cryo-EM images
In a message dated 5/27/2011 3:00:09 P.M. Eastern Daylight Time,
3dem-request at ncmir.ucsd.edu writes:
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Today's Topics:
1. Sucrose (Kimberley Cheng)
2. Re: Sucrose (Sacha De Carlo)
3. SV: [3dem] Sucrose (Kimberley Cheng)
----------------------------------------------------------------------
Message: 1
Date: Fri, 27 May 2011 08:24:05 +0000
From: Kimberley Cheng <Kimberley.Cheng at ki.se>
Subject: [3dem] Sucrose
To: "3dem at ncmir.ucsd.edu" <3dem at ncmir.ucsd.edu>
Message-ID:
<B491E5EE09A76243BE86D6F4F03B02222DEC2E5D at KIMSX04.user.ki.se>
Content-Type: text/plain; charset="us-ascii"
Dear all,
We are trying to analyze some samples that contain sucrose. Of course the
best would be to not have any sucrose at all, but in these cases we have no
choice since it's necessary for stability. My question is, does anybody
know the acceptable level of sucrose in vitrified specimens? Any literature
reference would be great.
All the best,
Kimberley
Dr. Kimberley Cheng
Karolinska Institutet, Department of Biosciences and Nutrition and
The Royal Institute of Technology, School of Technology and Health
Phone: +46-(0)8-608 92 17
Fax: +46-(0)8-608 92 90
E-mail: _Kimberley.Cheng at .ki.se_ (mailto:Kimberley.Cheng at .ki.se)
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