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Several years ago we had a similar problem, suddenly our usual
samples, imaged under the same conditions were bubbling at what used
to be the normal dose. After a while I complained and Quantifoil
told me they changed something in the cleaning procedure, probably
changed or reverted procedure and after that the new grids were fine
again. Perhaps that's the same problem that James referred to as
being solved quite a while back. I also recommend contacting the
manufacturer or the supplier.<br>
<br>
Good luck,<br>
Vladan <br>
<br>
--
<pre class="moz-signature" cols="72">Vladan Lucic, PhD
Max Planck Institute of Biochemistry
Dept of Molecular Structural Biology
Am Klopferspitz 18
82152 Martinsried, Germany
Pnone: +49 89 8578-2647
Fax: +49 89 8578-2643
Web: <a class="moz-txt-link-freetext" href="http://www.biochem.mpg.de/277447/15_ContentSynCompl">http://www.biochem.mpg.de/277447/15_ContentSynCompl</a></pre>
<div class="moz-cite-prefix">On 7/2/19 6:11 PM, Schwartz, Cindi
(NIH/NIAID) [E] wrote:<br>
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<p class="MsoPlainText">James,<o:p></o:p></p>
<p class="MsoPlainText"><o:p> </o:p></p>
<p class="MsoPlainText">Thanks for that insight. We do end up
with some direct from quanitfoil and some from the US supplier
(EMS in our case). But, we are having issues with both. Right
now, I tried a pretty extensive cleaning (sent to me
yesterday) protocol on these quantifoil supplied grids and I
have films after negative staining apoferritin. It's been a
disappointing morning looking at these grids. I will
definitely send these back to quantifoil and at least we know
that quantifoil says we can't remove the films once they've
been shipped so we should quit trying. The worst part is that
each grid is different, so in our hands checking randomly 5
out of 25 isn't enough. The two grids illustrated here are
from the same side of the same grid box chosen at random after
cleaning. That's why I wanted to bake on colloidal gold
fiducials on every grid so I could see if there was gold in
the holes, but alas, the colloidal gold doesn't seem to want
to stick to this plastic. I will also try this with different
kids of gold (maybe FAB-secondary anti-body gold will stick to
this plastic). <o:p></o:p></p>
<p class="MsoPlainText"><o:p> </o:p></p>
<p class="MsoPlainText">Here's the protocol I used from Dewight
Williams and Mariena Sylvestry-Ramos from when they were in
Phoebe Stewart's lab:<o:p></o:p></p>
<p class="MsoPlainText"><o:p> </o:p></p>
<p class="MsoPlainText">The key to dissolving the plastic on
quantifoils is to make the plastic flow away from the grid
surface. When you dissolve it and it does not migrate away it
just reabsorbs back on to the grid support surfaces. To get
this to happen you need to set up a thin layer chromatography
system, where you have a mobile phase of solvent moving up a
filter paper. We have used dichloraethane as the solvent in
combination with ethyl acetate. Pre-equilibrate a glass petri
dish with a saturated ethyl acetate soaked set of Whatman
filter papers with half a box of quantifoid grids, carbon side
up (plastic layer down at a slight incline (we used the grid
box to prop up one end. We also added a sharpie or other
permanent ink spot to the filter paper before starting to
confirm we had a mobile phase which would make the dye migrate
(I saw the dye migrate both times). Add to the bottom of the
slightly inclined petri dish a few mls of dichloroethane.
Place the lid on so the vapor will leave at the top of the
inclined petri dish (30 min). We then washed the grids in
chloroform (15 min) and acetone (15 min) the same way. In the
old days, we did this twice (I switched to a fresh clean glass
petri dish and fresh filter paper and repeated). Then baked in
a 60 C oven O/N. This morning I glow-discharged and then
negative stained apoferritin with Nano-Van.<o:p></o:p></p>
<p class="MsoPlainText"><o:p> </o:p></p>
<p class="MsoPlainText">Attached are the results. Disappointing
to say the least.<o:p></o:p></p>
<p class="MsoPlainText">Looks like I keep plugging away at it.
Thanks everyone for your ideas. BTW, other ideas I got were to
plasma clean the grids more, but I’m kind of scared to try
this as I don’t want to etch away all the carbon too!
<o:p></o:p></p>
<p class="MsoPlainText"><o:p> </o:p></p>
<p class="MsoPlainText">Cheers,<o:p></o:p></p>
<p class="MsoPlainText">Cindi L. Schwartz <o:p></o:p></p>
<p class="MsoPlainText"><o:p> </o:p></p>
<p class="MsoPlainText"><o:p> </o:p></p>
<p class="MsoPlainText">Electron Microscopist<o:p></o:p></p>
<p class="MsoPlainText">Rocky Mountain Labs/NIAID/NIH<o:p></o:p></p>
<p class="MsoPlainText">903 South 4th Street<o:p></o:p></p>
<p class="MsoPlainText">Hamilton, MT 59840<o:p></o:p></p>
<p class="MsoPlainText">406-363-9228<o:p></o:p></p>
<p class="MsoPlainText"><a class="moz-txt-link-abbreviated" href="mailto:Cindi.Schwartz@NIH.gov">Cindi.Schwartz@NIH.gov</a><o:p></o:p></p>
<p class="MsoPlainText"> Please remember our Earth before
printing this email <o:p></o:p></p>
<p class="MsoPlainText">*************************************************************************<o:p></o:p></p>
<p class="MsoPlainText">The information in this e-mail and any
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any statements made that are sender's own and not expressly
made on behalf of the NIAID by one of its representatives. <o:p></o:p></p>
<p class="MsoPlainText">*************************************************************************<o:p></o:p></p>
<p class="MsoPlainText"><o:p> </o:p></p>
<p class="MsoPlainText">-----Original Message-----<br>
From: Conway, James Frederick <a class="moz-txt-link-rfc2396E" href="mailto:James.Conway@pitt.edu"><James.Conway@pitt.edu></a> <br>
Sent: Tuesday, July 2, 2019 8:26 AM<br>
To: <a class="moz-txt-link-abbreviated" href="mailto:3dem@ncmir.ucsd.edu">3dem@ncmir.ucsd.edu</a><br>
Cc: Schwartz, Cindi (NIH/NIAID) [E]
<a class="moz-txt-link-rfc2396E" href="mailto:cindi.schwartz@nih.gov"><cindi.schwartz@nih.gov></a><br>
Subject: Re: [3dem] Help! Plastic films on my holey grids are
KILLING ME</p>
<p class="MsoPlainText"><o:p> </o:p></p>
<p class="MsoPlainText">It was my understanding that Quantifoil
solved this problem quite a while back, but nonetheless we
kept receiving plastic-contaminated grids from one US supplier
for some time after. When I contacted Quantifoil, they said
that removing the plastic after the factory was near
impossible, and when I told them the serial number of the grid
boxes, they disavowed knowledge of them. My dark suspicion is
that sending them back to the supplier just put them in the
pipeline for someone else to receive, but that would be rank
speculation. However, buying directly from Quantifoil has
resulted in near perfect grids for over 5 years now. I suggest
sending the bad batches of grids back for a refund, and if you
are not already dealing directly with Quantifoil, try doing so
or with a factory-recommended local supplier. If these grids
are from Quantifoil, then certainly complain, send them back -
it shouldn't be your time and money to fix up the grids. In my
experience C-flats behave differently and (in my hands)
inconsistently, but I understand there was a period of
manufacturing issues that are now resolved and I look forward
to trying them again.<o:p></o:p></p>
<p class="MsoPlainText"><o:p> </o:p></p>
<p class="MsoPlainText">James Conway<o:p></o:p></p>
<p class="MsoPlainText">----------------------------------------------------------------------<o:p></o:p></p>
<p class="MsoPlainText">James Conway, PhD.,<o:p></o:p></p>
<p class="MsoPlainText">Professor, Department of Structural
Biology Director, Molecular Biophysics and Structural Biology
Graduate Program University of Pittsburgh School of Medicine
Biomedical Science Tower 3, Room 2047<o:p></o:p></p>
<p class="MsoPlainText">3501 5th Ave<o:p></o:p></p>
<p class="MsoPlainText">Pittsburgh, PA 15213<o:p></o:p></p>
<p class="MsoPlainText">U.S.A.<o:p></o:p></p>
<p class="MsoPlainText">Phone: +1-412-383-9847<o:p></o:p></p>
<p class="MsoPlainText">Fax: +1-412-648-8998<o:p></o:p></p>
<p class="MsoPlainText">Email: <a href="mailto:jxc100@pitt.edu"
moz-do-not-send="true"><span
style="color:windowtext;text-decoration:none">jxc100@pitt.edu</span></a><o:p></o:p></p>
<p class="MsoPlainText">Web: <a
href="http://www.structbio.pitt.edu/index.php/12-faculty/18-james-conway"
moz-do-not-send="true">
<span style="color:windowtext;text-decoration:none">http://www.structbio.pitt.edu/index.php/12-faculty/18-james-conway</span></a>
<o:p></o:p></p>
<p class="MsoPlainText">----------------------------------------------------------------------<o:p></o:p></p>
<p class="MsoPlainText"><o:p> </o:p></p>
<p class="MsoPlainText"><o:p> </o:p></p>
<p class="MsoPlainText"><o:p> </o:p></p>
<p class="MsoPlainText"><o:p> </o:p></p>
<p class="MsoPlainText"><o:p> </o:p></p>
<p class="MsoPlainText"><o:p> </o:p></p>
<p class="MsoPlainText"><o:p> </o:p></p>
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