SV: [3dem] Re: 3dem Digest, Vol 45, Issue 16

[Kimberley Cheng]

Hello Dave,

Thank you so much for your feedback!! It taught me a lot.

Your "Spin Column" method sounds very interesting and if you don't mind I would be very happy to receive a more detailed method description. One question, does this method work for all kinds of samples? I mean, are there any restrictions?

Thanks again!

All the best,
Kimberley
________________________________
Från: 3dem-bounces at ncmir.ucsd.edu [3dem-bounces at ncmir.ucsd.edu] för DocDave50 at aol.com [DocDave50 at aol.com]
Skickat: den 28 maj 2011 17:49
Till: 3dem at ncmir.ucsd.edu
Ämne: [3dem] Re: 3dem Digest, Vol 45, Issue 16

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 almost 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)


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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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