[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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