<html xmlns:o="urn:schemas-microsoft-com:office:office" xmlns:w="urn:schemas-microsoft-com:office:word" xmlns:m="http://schemas.microsoft.com/office/2004/12/omml" xmlns="http://www.w3.org/TR/REC-html40">
<head>
<meta http-equiv="Content-Type" content="text/html; charset=utf-8">
<meta name="Generator" content="Microsoft Word 15 (filtered medium)">
<style><!--
/* Font Definitions */
@font-face
{font-family:"Cambria Math";
panose-1:2 4 5 3 5 4 6 3 2 4;}
@font-face
{font-family:Calibri;
panose-1:2 15 5 2 2 2 4 3 2 4;}
/* Style Definitions */
p.MsoNormal, li.MsoNormal, div.MsoNormal
{margin:0cm;
margin-bottom:.0001pt;
font-size:11.0pt;
font-family:"Calibri",sans-serif;}
span.E-MailFormatvorlage18
{mso-style-type:personal-reply;
font-family:"Calibri",sans-serif;
color:windowtext;}
.MsoChpDefault
{mso-style-type:export-only;
font-size:10.0pt;}
@page WordSection1
{size:612.0pt 792.0pt;
margin:70.85pt 70.85pt 2.0cm 70.85pt;}
div.WordSection1
{page:WordSection1;}
--></style>
</head>
<body lang="DE" link="#0563C1" vlink="#954F72">
<div class="WordSection1">
<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 lang="EN-US" style="mso-fareast-language: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 lang="EN-US" style="mso-fareast-language:EN-US"><o:p> </o:p></span></p>
<p class="MsoNormal"><span lang="EN-US" style="mso-fareast-language: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 lang="EN-US" style="color:black">------------------------------------------------------------------------------------------------<o:p></o:p></span></p>
<p class="MsoNormal"><b><span lang="EN-US" style="color:black">Magdalena Schacherl, PhD<o:p></o:p></span></b></p>
<p class="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"><o:p></o:p></span></i></p>
<p class="MsoNormal"><span lang="EN-US" style="color:black">Charité - Universitätsmedizin Berlin<o:p></o:p></span></p>
<p class="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<o:p></o:p></span></p>
<p class="MsoNormal"><span lang="EN-US" style="color:black">Internal address: Virchowweg 6<o:p></o:p></span></p>
<p class="MsoNormal"><span lang="EN-US" style="color:black">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"><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$"><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 lang="EN-US" style="mso-fareast-language:EN-US"><o:p> </o:p></span></p>
<p class="MsoNormal"><span lang="EN-US" style="mso-fareast-language: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 <3dem-bounces@ncmir.ucsd.edu> im Auftrag von Umar Farook <umarfarook12@gmail.com><br>
<b>Datum: </b>Donnerstag, 4. Juni 2020 um 21:25<br>
<b>An: </b>"3dem@ncmir.ucsd.edu" <3dem@ncmir.ucsd.edu><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>
</body>
</html>