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<p>Hi Umar,</p>
<p>Or if you know already that your protein is happy on a surface
you might want to give graphene or graphene oxide grids a try. <br>
</p>
<p>Best,</p>
<p>Felipe<br>
</p>
<div class="moz-cite-prefix">On 04.06.20 21:57, Schacherl, Magdalena
wrote:<br>
</div>
<blockquote type="cite"
cite="mid:FD1B8CF0-DDDB-4F85-AC07-0C44304C02CF@charite.de">
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<p class="MsoNormal"><span style="mso-fareast-language:EN-US">Hi
Umar,<o:p></o:p></span></p>
<p class="MsoNormal"><span style="mso-fareast-language:EN-US"><o:p> </o:p></span></p>
<p class="MsoNormal"><span style="mso-fareast-language:EN-US"
lang="EN-US">I agree with Jay, that using detergents or
surfactants (octyl glucoside 0.1% final conc. or fluorinated
octyl maltoside 0.0125% final conc.) might help you. The
detergents repel the protein from the water-air interface.
Be aware of the fact, that they make the ice thicker and you
need a much higher concentration of protein as compared to
blotting without detergents.
<o:p></o:p></span></p>
<p class="MsoNormal"><span style="mso-fareast-language:EN-US"
lang="EN-US"><o:p> </o:p></span></p>
<p class="MsoNormal"><span style="mso-fareast-language:EN-US"
lang="EN-US">If blotting per se (the force applied by the
contact with filter paper) is the problem, you could try to
fix the hexamer with mild glutaraldehyde treatment prior to
blotting. We occasionally use </span><span lang="EN-US">0.5%
(v/v) glutaraldehyde (EM grade) and incubate for 10 min at 4
°C, then immediately blot and plunge freeze.<o:p></o:p></span></p>
<p class="MsoNormal"><span lang="EN-US"><o:p> </o:p></span></p>
<p class="MsoNormal"><span lang="EN-US">If everything fails,
then you might want to try blot-free techniques to apply
your protein to grids (e.g. spotting or writing) – there are
commercial machines around like Chameleon or VitroJet, or
you try to spray your protein (see S. Muench lab or J. Frank
lab, and others).<o:p></o:p></span></p>
<p class="MsoNormal"><span lang="EN-US"><o:p> </o:p></span></p>
<p class="MsoNormal"><span lang="EN-US">Best regards, <o:p></o:p></span></p>
<p class="MsoNormal"><span lang="EN-US"><o:p> </o:p></span></p>
<p class="MsoNormal"><span lang="EN-US">Magdalena<o:p></o:p></span></p>
<p class="MsoNormal"><span lang="EN-US"><o:p> </o:p></span></p>
<p class="MsoNormal"><o:p> </o:p></p>
<p class="MsoNormal"><span style="color:black" lang="EN-US">------------------------------------------------------------------------------------------------<o:p></o:p></span></p>
<p class="MsoNormal"><b><span style="color:black" lang="EN-US">Magdalena
Schacherl, PhD<o:p></o:p></span></b></p>
<p class="MsoNormal"><b><i><span style="color:black"
lang="EN-US">Group Leader Structural Enzymology</span></i></b><i><span
style="color:black" lang="EN-US"><o:p></o:p></span></i></p>
<p class="MsoNormal"><span style="color:black" lang="EN-US">Charité
- Universitätsmedizin Berlin<o:p></o:p></span></p>
<p class="MsoNormal"><span style="color:black" lang="EN">Institute
of Medical Physics and Biophysics
</span><span style="color:black" lang="EN-US"> | Charitéplatz
1 | 10117 Berlin<o:p></o:p></span></p>
<p class="MsoNormal"><span style="color:black" lang="EN-US">Internal
address: Virchowweg 6<o:p></o:p></span></p>
<p class="MsoNormal"><span style="color:black" lang="EN-US">Room
03.322, level 3<o:p></o:p></span></p>
<p class="MsoNormal"><span style="color:black">T: +49 30 450
524196 | F: +49 30 450 524952<o:p></o:p></span></p>
<p class="MsoNormal"><a
href="mailto:magdalena.schacherl@charite.de"
moz-do-not-send="true"><span style="color:black">magdalena.schacherl@charite.de</span></a><span
style="color:black"><o:p></o:p></span></p>
<p class="MsoNormal"><span style="color:black"> <o:p></o:p></span></p>
<p class="MsoNormal"><a
href="https://urldefense.com/v3/__https://biophysik.charite.de/en/research/structural_enzymology_schacherl_lab/__;!!Mih3wA!RCBOFKYqP30RiDg5ED5tufy2vsc_-ByfjO5mvQAv7w-7fIIqLsbhR2y2YIoqQEZRjQ$"
moz-do-not-send="true"><span style="color:#4472C4">Website
Schacherl lab</span></a><span style="color:#4472C4"><o:p></o:p></span></p>
<p class="MsoNormal"><o:p> </o:p></p>
<p class="MsoNormal"><span lang="EN-US"><o:p> </o:p></span></p>
<p class="MsoNormal"><span style="mso-fareast-language:EN-US"
lang="EN-US"><o:p> </o:p></span></p>
<p class="MsoNormal"><span style="mso-fareast-language:EN-US"
lang="EN-US"><o:p> </o:p></span></p>
<div style="border:none;border-top:solid #B5C4DF
1.0pt;padding:3.0pt 0cm 0cm 0cm">
<p class="MsoNormal"><b><span
style="font-size:12.0pt;color:black">Von: </span></b><span
style="font-size:12.0pt;color:black">3dem
<a class="moz-txt-link-rfc2396E" href="mailto:3dem-bounces@ncmir.ucsd.edu"><3dem-bounces@ncmir.ucsd.edu></a> im Auftrag von Umar
Farook <a class="moz-txt-link-rfc2396E" href="mailto:umarfarook12@gmail.com"><umarfarook12@gmail.com></a><br>
<b>Datum: </b>Donnerstag, 4. Juni 2020 um 21:25<br>
<b>An: </b><a class="moz-txt-link-rfc2396E" href="mailto:3dem@ncmir.ucsd.edu">"3dem@ncmir.ucsd.edu"</a>
<a class="moz-txt-link-rfc2396E" href="mailto:3dem@ncmir.ucsd.edu"><3dem@ncmir.ucsd.edu></a><br>
<b>Betreff: </b>[ext] [3dem] Hexamer dissociate while
freezing Cryo-EM grids<o:p></o:p></span></p>
</div>
<div>
<p class="MsoNormal"><o:p> </o:p></p>
</div>
<div>
<p class="MsoNormal">Dear All, <o:p></o:p></p>
<div>
<p class="MsoNormal"><o:p> </o:p></p>
</div>
<div>
<p class="MsoNormal">I would like to ask the community for
suggestions on how to overcome the issue of hexamer
dissociating while freezing Cryo-EM grids, the negative
staining seems to be perfect hexamer at 0.1 mg/ml
concentration. But the sample seems to fall apart at 1.5
mg/ml in Cryo freezing, please advise, thank you.<o:p></o:p></p>
</div>
<div>
<p class="MsoNormal"><o:p> </o:p></p>
</div>
<div>
<p class="MsoNormal">Best Regards,<o:p></o:p></p>
</div>
<div>
<p class="MsoNormal">Umar<o:p></o:p></p>
</div>
</div>
</div>
<br>
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<pre class="moz-quote-pre" wrap="">_______________________________________________
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</pre>
</blockquote>
<pre class="moz-signature" cols="72">--
Felipe Merino,
Max Planck Institute for Developmental Biology
Department of Protein Evolution
Max-Planck-Ring 5
72076 Tuebingen
Phone: +49 7071 601 1351
ORCID: <a class="moz-txt-link-freetext" href="https://urldefense.com/v3/__https://orcid.org/0000-0003-4166-8747__;!!Mih3wA!Wovm-gVQiGlBAGs2Q9dEbwbScSTOZHyMZnEfw6hf5aactNdJuD44gYtwQi4DHBc_cA$">https://orcid.org/0000-0003-4166-8747</a>
Publons: <a class="moz-txt-link-freetext" href="https://urldefense.com/v3/__https://publons.com/a/1305699/__;!!Mih3wA!Wovm-gVQiGlBAGs2Q9dEbwbScSTOZHyMZnEfw6hf5aactNdJuD44gYtwQi75IBpG2Q$">https://publons.com/a/1305699/</a>
Researchgate: <a class="moz-txt-link-freetext" href="https://urldefense.com/v3/__https://www.researchgate.net/profile/Felipe_Merino__;!!Mih3wA!Wovm-gVQiGlBAGs2Q9dEbwbScSTOZHyMZnEfw6hf5aactNdJuD44gYtwQi7Xv37Ung$">https://www.researchgate.net/profile/Felipe_Merino</a></pre>
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