<html>
<head>
<meta http-equiv="Content-Type" content="text/html; charset=Windows-1252">
</head>
<body>
<div dir="ltr">
<div></div>
<div data-ogsc="" style="">
<div>If your proteins complexes are too small and cannot get the enough contrast with Graphene oxide or in thicker ice then you can give a shot with strepavidine Monolayer.</div>
<div dir="ltr"><br>
</div>
<div><br>
</div>
<div class="ms-outlook-ios-signature" id="ms-outlook-mobile-signature">
<div style="direction: ltr;">With kind regards,</div>
<div style="direction: ltr;">Jay</div>
</div>
</div>
</div>
<hr style="display:inline-block;width:98%" tabindex="-1">
<div id="divRplyFwdMsg" dir="ltr"><font face="Calibri, sans-serif" style="font-size:11pt" color="#000000"><b>From:</b> 3dem <3dem-bounces@ncmir.ucsd.edu> on behalf of Felipe Merino <felipe.merino@tuebingen.mpg.de><br>
<b>Sent:</b> Thursday, June 4, 2020 4:12:28 PM<br>
<b>To:</b> 3dem@ncmir.ucsd.edu <3dem@ncmir.ucsd.edu><br>
<b>Subject:</b> Re: [3dem] [ext] Hexamer dissociate while freezing Cryo-EM grids</font>
<div> </div>
</div>
<div>
<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="x_moz-cite-prefix">On 04.06.20 21:57, Schacherl, Magdalena wrote:<br>
</div>
<blockquote type="cite">
<meta name="Generator" content="Microsoft Word 15 (filtered
medium)">
<style>
<!--
@font-face
{font-family:"Cambria Math"}
@font-face
{font-family:Calibri}
p.x_MsoNormal, li.x_MsoNormal, div.x_MsoNormal
{margin:0cm;
margin-bottom:.0001pt;
font-size:11.0pt;
font-family:"Calibri",sans-serif}
span.x_E-MailFormatvorlage18
{font-family:"Calibri",sans-serif;
color:windowtext}
.x_MsoChpDefault
{font-size:10.0pt}
@page WordSection1
{margin:70.85pt 70.85pt 2.0cm 70.85pt}
div.x_WordSection1
{}
-->
</style>
<div class="x_WordSection1">
<p class="x_MsoNormal"><span style="">Hi Umar,</span></p>
<p class="x_MsoNormal"><span style=""> </span></p>
<p class="x_MsoNormal"><span lang="EN-US" style="">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.
</span></p>
<p class="x_MsoNormal"><span lang="EN-US" style=""> </span></p>
<p class="x_MsoNormal"><span lang="EN-US" style="">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.</span></p>
<p class="x_MsoNormal"><span lang="EN-US"> </span></p>
<p class="x_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).</span></p>
<p class="x_MsoNormal"><span lang="EN-US"> </span></p>
<p class="x_MsoNormal"><span lang="EN-US">Best regards, </span></p>
<p class="x_MsoNormal"><span lang="EN-US"> </span></p>
<p class="x_MsoNormal"><span lang="EN-US">Magdalena</span></p>
<p class="x_MsoNormal"><span lang="EN-US"> </span></p>
<p class="x_MsoNormal"> </p>
<p class="x_MsoNormal"><span lang="EN-US" style="color:black">------------------------------------------------------------------------------------------------</span></p>
<p class="x_MsoNormal"><b><span lang="EN-US" style="color:black">Magdalena Schacherl, PhD</span></b></p>
<p class="x_MsoNormal"><b><i><span lang="EN-US" style="color:black">Group Leader Structural Enzymology</span></i></b><i><span lang="EN-US" style="color:black"></span></i></p>
<p class="x_MsoNormal"><span lang="EN-US" style="color:black">Charité - Universitätsmedizin Berlin</span></p>
<p class="x_MsoNormal"><span lang="EN" style="color:black">Institute of Medical Physics and Biophysics
</span><span lang="EN-US" style="color:black"> | Charitéplatz 1 | 10117 Berlin</span></p>
<p class="x_MsoNormal"><span lang="EN-US" style="color:black">Internal address: Virchowweg 6</span></p>
<p class="x_MsoNormal"><span lang="EN-US" style="color:black">Room 03.322, level 3</span></p>
<p class="x_MsoNormal"><span style="color:black">T: +49 30 450 524196 | F: +49 30 450 524952</span></p>
<p class="x_MsoNormal"><a href="mailto:magdalena.schacherl@charite.de"><span style="color:black">magdalena.schacherl@charite.de</span></a><span style="color:black"></span></p>
<p class="x_MsoNormal"><span style="color:black"> </span></p>
<p class="x_MsoNormal"><a href="https://urldefense.com/v3/__https://biophysik.charite.de/en/research/structural_enzymology_schacherl_lab/__;!!Mih3wA!RCBOFKYqP30RiDg5ED5tufy2vsc_-ByfjO5mvQAv7w-7fIIqLsbhR2y2YIoqQEZRjQ$"><span style="color:#4472C4">Website Schacherl
lab</span></a><span style="color:#4472C4"></span></p>
<p class="x_MsoNormal"> </p>
<p class="x_MsoNormal"><span lang="EN-US"> </span></p>
<p class="x_MsoNormal"><span lang="EN-US" style=""> </span></p>
<p class="x_MsoNormal"><span lang="EN-US" style=""> </span></p>
<div style="border:none; border-top:solid #B5C4DF
1.0pt; padding:3.0pt 0cm 0cm 0cm">
<p class="x_MsoNormal"><b><span style="font-size:12.0pt; color:black">Von: </span>
</b><span style="font-size:12.0pt; color:black">3dem <a class="x_moz-txt-link-rfc2396E" href="mailto:3dem-bounces@ncmir.ucsd.edu">
<3dem-bounces@ncmir.ucsd.edu></a> im Auftrag von Umar Farook <a class="x_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="x_moz-txt-link-rfc2396E" href="mailto:3dem@ncmir.ucsd.edu">"3dem@ncmir.ucsd.edu"</a>
<a class="x_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</span></p>
</div>
<div>
<p class="x_MsoNormal"> </p>
</div>
<div>
<p class="x_MsoNormal">Dear All, </p>
<div>
<p class="x_MsoNormal"> </p>
</div>
<div>
<p class="x_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.</p>
</div>
<div>
<p class="x_MsoNormal"> </p>
</div>
<div>
<p class="x_MsoNormal">Best Regards,</p>
</div>
<div>
<p class="x_MsoNormal">Umar</p>
</div>
</div>
</div>
<br>
<fieldset class="x_mimeAttachmentHeader"></fieldset>
<pre class="x_moz-quote-pre">_______________________________________________
3dem mailing list
<a class="x_moz-txt-link-abbreviated" href="mailto:3dem@ncmir.ucsd.edu">3dem@ncmir.ucsd.edu</a>
<a class="x_moz-txt-link-freetext" href="https://urldefense.com/v3/__https://mail.ncmir.ucsd.edu/mailman/listinfo/3dem__;!!PhOWcWs!k48unNWj5DJOh_kn1gA95BSAKgh7Jc1qqGA83iQPUT2OmrJVyx2MRdVMngs8$">https://mail.ncmir.ucsd.edu/mailman/listinfo/3dem</a>
</pre>
</blockquote>
<pre class="x_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="x_moz-txt-link-freetext" href="https://urldefense.com/v3/__https://orcid.org/0000-0003-4166-8747__;!!Mih3wA!Wovm-gVQiGlBAGs2Q9dEbwbScSTOZHyMZnEfw6hf5aactNdJuD44gYtwQi4DHBc_cA$">https://orcid.org/0000-0003-4166-8747</a>
Publons: <a class="x_moz-txt-link-freetext" href="https://urldefense.com/v3/__https://publons.com/a/1305699/__;!!Mih3wA!Wovm-gVQiGlBAGs2Q9dEbwbScSTOZHyMZnEfw6hf5aactNdJuD44gYtwQi75IBpG2Q$">https://publons.com/a/1305699/</a>
Researchgate: <a class="x_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>
</div>
</body>
</html>