[3dem] 3dem Digest, Vol 79, Issue 18
DocDave50 at aol.com
DocDave50 at aol.com
Wed Mar 19 12:36:10 PDT 2014
"...Dear colleagues,
I work with HIV particles and (at least in my samples), they like the
carbon film on the grids much better than the holes, but for me this is a big
problem, as, even using high titer preps, they rarely go to the holes,
resulting in less-than-perfect tomograms. Has anyone else had this problem? What
did you do to solve it?
My particles are resusspended in PBS (with gold fiducials) at the time of
freezing.
Best,
Luiza..."
Luiza,
The principle difficulty, as mentioned by some has to do with carbon
surface charge distribution. As you know, the longer a carbon film sits, the
more hydrophobic the surface becomes. Ben's comment about shorter glow
discharge times does not make the grid more hydrophilic but, rather, does not
alter the hydrophilicity much. Hence, his suggestion of a very short glow
discharge time would mean that the surface would still be quite hydrophobic and
not wet. The problem resides in the holes themselves. The edges of the
holes have a finite thickness depending on the grid source and, even with
significant glow discharge, you are not altering the hydrophobicity of the hole
edges. We noticed this when looking at lipid vesicles which were freeze
quenched. Without fail, the vesicles always went to the hole edge and were
unusable for examination.
What is necessary for success here is that the hole is 'wetable' while
the carbon surface tends not to be. Both Ben and Qiu Xing have good ideas.
Here is another...or at least one more...depends on how long I think about the
situation. The longer I think, more options begin to emerge. A key to glow
discharging a grid is that only the side you which is up actually 'sees'
the plasma and is, therefore, charged. As I stated, in our experience, even
glow discharging both sides of a holey carbon grid gave us lipid vesicles
which liked the hole edges (Qiu Xing will concur since we did these
together at one point). Additionally, ice thickness in the hole needs to be
non-convex in nature such that the ice is thinner in the center than at the edges
of the hole.
So, here is one way to possibly defeat the issue that you are dealing
with, in essence, you are 'pre-wetting' your grid prior to sample application.
Try not glow discharging your grids at all or using Ben's suggestion of
just a very short time. Just prior to applying your sample, pre-wet your grid
by applying 3 μl of your PBS buffer containing a very dilute detergent
concentration...something well below it's CMC (by say 100 fold) and at a
concentration in which detergent desorption from the carbon hydrophobic surface
is very slow because the detergent binds very tightly to the hole...and the
carbon surface, of course. Carefully blot the grid surface and then add
your virus sample and freeze quench right away. The low detergent
concentration effectively makes the hole edges wetable and blotting wicks/blots away
the detergent solution on the carbon surface, mimicking Ben's suggestion.
If your sample application and freeze quenching occurs quickly after
blotting away excess PBS/ Detergent, the detergent molecules forming an
amphipathic coating on the hole surface should not have sufficient time to desorb
from the carbon and into your HIV envelope coat and, as such, allow you to
form a nice and even thickness of ice in the holes.
Just a thought. I have tried this with success but not with your system
and/or conditions.
With my Best Wishes,
David
David W. Chester, Ph.D.
Founder and Research Consultant
Analytical BioConsulting, Inc
.
64 Corrigan Avenue
Meriden, CT. 06268
Phone: (203) 213-4167
Email: docdave50 at aol.com
In a message dated 3/14/2014 12:20:02 P.M. Eastern Daylight Time,
3dem-request at ncmir.ucsd.edu writes:
Send 3dem mailing list submissions to
3dem at ncmir.ucsd.edu
To subscribe or unsubscribe via the World Wide Web, visit
https://mail.ncmir.ucsd.edu/mailman/listinfo/3dem
or, via email, send a message with subject or body 'help' to
3dem-request at ncmir.ucsd.edu
You can reach the person managing the list at
3dem-owner at ncmir.ucsd.edu
When replying, please edit your Subject line so it is more specific
than "Re: Contents of 3dem digest..."
Today's Topics:
3. Re: Virus on Carbon (Qiu-Xing Jiang)
4. Re: Virus on Carbon (Ben Hankamer)
Message: 3
Date: Fri, 14 Mar 2014 11:54:53 +0000
From: Qiu-Xing Jiang <Qiu-Xing.Jiang at UTSouthwestern.edu>
To: C.J. <biocjh at gmail.com>, 3DEM Mailing List <3dem at ncmir.ucsd.edu>
Subject: Re: [3dem] Virus on Carbon
Message-ID:
<6306D158E60520409F67980750A5AA618AF683 at swmsmail5.swmed.org>
Content-Type: text/plain; charset="gb2312"
We recently developed chemically modified carbon films. Protein G on C
films may help select viral particles through an antibody. It could allow the
concentration ~0.1 - 1% of what usually used, and may be useful. Marc
Llaguno from my lab is on the Tahoe meeting presenting this. If you are there,
please go talk to him. We could send some grids to you. Best luck,
__________________________________________________
Qiu-Xing Jiang
Department of Cell Biology
UT Southwestern Medical Center
Rm NL5.140B, MC9039
6000 Harry Hines Blvd
Dallas, TX 75390, USA
phone: 214-633-1940 Fax 214-648-5814
Admin Assistant: Ms. Demyia Pridgen (phone 214-633-1948)
________________________________________
From: 3dem-bounces at ncmir.ucsd.edu [3dem-bounces at ncmir.ucsd.edu] on behalf
of C.J. [biocjh at gmail.com]
Sent: Thursday, March 13, 2014 11:23 PM
To: 3DEM Mailing List
Subject: Re: [3dem] Virus on Carbon
I also came accross problems of something like Luiza's. My sample is a
macromolecule.
Xinghong, whether your method could solve my problem? Thanks.
2014-03-14 12:16 GMT+08:00 Xinghong Dai
<bestdz at gmail.com<mailto:bestdz at gmail.com>>:
Hi Luiza,
Maybe you can try to make the ice a little bit thicker to see if that will
make any difference. They theory is that, if the ice is too thin, big
particles will not stay in the hole due to size exclusion effect. I had similar
situation when working with big viruses.
Good luck!
Xinghong
Message: 4
Date: Thu, 13 Mar 2014 22:51:11 -0700
From: Ben Hankamer <b.hankamer at uq.edu.au>
To: Luiza Mendon?a <luiza at caltech.edu>
Cc: 3DEM Mailing List <3dem at ncmir.ucsd.edu>
Subject: Re: [3dem] Virus on Carbon
Message-ID: <687303DB-348A-45B1-AA5A-6734FF77E486 at uq.edu.au>
Content-Type: text/plain; charset=utf-8
Hi Luiza
We have had similar problems with a number of proteins. What worked for us
in some cases was to reduce the glow discharge time. It will depend on
your set up but try down to 1 sec.
The rational was to make the carbon sufficiently hydrophilic to allow the
ice sheet to form properly in the holes of the holey carbon film, but to
minimize electrostatic attraction of the proteins to the carbon.
Ben
> On 13 Mar 2014, at 4:42 pm, "Luiza Mendon?a" <luiza at caltech.edu> wrote:
>
> Dear colleagues,
>
> I work with HIV particles and (at least in my samples), they like the
carbon film on the grids much better than the holes, but for me this is a big
problem, as, even using high titer preps, they rarely go to the holes,
resulting in less-than-perfect tomograms. Has anyone else had this problem?
What did you do to solve it?
> My particles are resusspended in PBS (with gold fiducials) at the time
of freezing.
>
> Best,
> Luiza.
-------------- next part --------------
An HTML attachment was scrubbed...
URL: <https://mail.ncmir.ucsd.edu/mailman/private/3dem/attachments/20140319/c24709d5/attachment.html>
More information about the 3dem
mailing list