<div dir="ltr"><div>Hi Umar,</div><div><br></div><div>Another option is use GO grid then you can sty at the same concentration (+/-) as the NS and you also don't have the air/water interface.</div><div><br></div><div>Best,<br></div></div><br><div class="gmail_quote"><div dir="ltr" class="gmail_attr">On Thu, Jun 4, 2020 at 2:57 PM <<a href="mailto:3dem-request@ncmir.ucsd.edu">3dem-request@ncmir.ucsd.edu</a>> wrote:<br></div><blockquote class="gmail_quote" style="margin:0px 0px 0px 0.8ex;border-left:1px solid rgb(204,204,204);padding-left:1ex">Send 3dem mailing list submissions to<br>
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Today's Topics:<br>
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1. Hexamer dissociate while freezing Cryo-EM grids (Umar Farook)<br>
2. Re: Hexamer dissociate while freezing Cryo-EM grids (Jay Rai)<br>
3. Re: [ext] Hexamer dissociate while freezing Cryo-EM grids<br>
(Schacherl, Magdalena)<br>
<br>
<br>
----------------------------------------------------------------------<br>
<br>
Message: 1<br>
Date: Thu, 4 Jun 2020 22:24:44 +0300<br>
From: Umar Farook <<a href="mailto:umarfarook12@gmail.com" target="_blank">umarfarook12@gmail.com</a>><br>
To: <a href="mailto:3dem@ncmir.ucsd.edu" target="_blank">3dem@ncmir.ucsd.edu</a><br>
Subject: [3dem] Hexamer dissociate while freezing Cryo-EM grids<br>
Message-ID:<br>
<CAFGvJnbAJwN6N-SiavXc1uH4Dh=<a href="mailto:ESCP7C7oWC9ZXdscj9et_pA@mail.gmail.com" target="_blank">ESCP7C7oWC9ZXdscj9et_pA@mail.gmail.com</a>><br>
Content-Type: text/plain; charset="utf-8"<br>
<br>
Dear All,<br>
<br>
I would like to ask the community for suggestions on how to overcome the<br>
issue of hexamer dissociating while freezing Cryo-EM grids, the negative<br>
staining seems to be perfect hexamer at 0.1 mg/ml concentration. But the<br>
sample seems to fall apart at 1.5 mg/ml in Cryo freezing, please advise,<br>
thank you.<br>
<br>
Best Regards,<br>
Umar<br>
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<br>
Message: 2<br>
Date: Thu, 4 Jun 2020 19:26:47 +0000<br>
From: Jay Rai <<a href="mailto:jrai@fsu.edu" target="_blank">jrai@fsu.edu</a>><br>
To: Umar Farook <<a href="mailto:umarfarook12@gmail.com" target="_blank">umarfarook12@gmail.com</a>>, "<a href="mailto:3dem@ncmir.ucsd.edu" target="_blank">3dem@ncmir.ucsd.edu</a>"<br>
<<a href="mailto:3dem@ncmir.ucsd.edu" target="_blank">3dem@ncmir.ucsd.edu</a>><br>
Subject: Re: [3dem] Hexamer dissociate while freezing Cryo-EM grids<br>
Message-ID:<br>
<<a href="mailto:DM6PR02MB5276D19D53787E0F8196B1B1D0890@DM6PR02MB5276.namprd02.prod.outlook.com" target="_blank">DM6PR02MB5276D19D53787E0F8196B1B1D0890@DM6PR02MB5276.namprd02.prod.outlook.com</a>><br>
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Content-Type: text/plain; charset="us-ascii"<br>
<br>
Your sample might destroy by air-water interference.<br>
Remedy- try using detergent.<br>
Jay<br>
<br>
With kind regards,<br>
Jay<br>
________________________________<br>
From: 3dem <<a href="mailto:3dem-bounces@ncmir.ucsd.edu" target="_blank">3dem-bounces@ncmir.ucsd.edu</a>> on behalf of Umar Farook <<a href="mailto:umarfarook12@gmail.com" target="_blank">umarfarook12@gmail.com</a>><br>
Sent: Thursday, June 4, 2020 3:24:44 PM<br>
To: <a href="mailto:3dem@ncmir.ucsd.edu" target="_blank">3dem@ncmir.ucsd.edu</a> <<a href="mailto:3dem@ncmir.ucsd.edu" target="_blank">3dem@ncmir.ucsd.edu</a>><br>
Subject: [3dem] Hexamer dissociate while freezing Cryo-EM grids<br>
<br>
Dear All,<br>
<br>
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.<br>
<br>
Best Regards,<br>
Umar<br>
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------------------------------<br>
<br>
Message: 3<br>
Date: Thu, 4 Jun 2020 19:57:14 +0000<br>
From: "Schacherl, Magdalena" <<a href="mailto:magdalena.schacherl@charite.de" target="_blank">magdalena.schacherl@charite.de</a>><br>
To: Umar Farook <<a href="mailto:umarfarook12@gmail.com" target="_blank">umarfarook12@gmail.com</a>><br>
Cc: "<a href="mailto:3dem@ncmir.ucsd.edu" target="_blank">3dem@ncmir.ucsd.edu</a>" <<a href="mailto:3dem@ncmir.ucsd.edu" target="_blank">3dem@ncmir.ucsd.edu</a>><br>
Subject: Re: [3dem] [ext] Hexamer dissociate while freezing Cryo-EM<br>
grids<br>
Message-ID: <<a href="mailto:FD1B8CF0-DDDB-4F85-AC07-0C44304C02CF@charite.de" target="_blank">FD1B8CF0-DDDB-4F85-AC07-0C44304C02CF@charite.de</a>><br>
Content-Type: text/plain; charset="utf-8"<br>
<br>
Hi Umar,<br>
<br>
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.<br>
<br>
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 0.5% (v/v) glutaraldehyde (EM grade) and incubate for 10 min at 4 ?C, then immediately blot and plunge freeze.<br>
<br>
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).<br>
<br>
Best regards,<br>
<br>
Magdalena<br>
<br>
<br>
------------------------------------------------------------------------------------------------<br>
Magdalena Schacherl, PhD<br>
Group Leader Structural Enzymology<br>
Charit? - Universit?tsmedizin Berlin<br>
Institute of Medical Physics and Biophysics | Charit?platz 1 | 10117 Berlin<br>
Internal address: Virchowweg 6<br>
Room 03.322, level 3<br>
T: +49 30 450 524196 | F: +49 30 450 524952<br>
<a href="mailto:magdalena.schacherl@charite.de" target="_blank">magdalena.schacherl@charite.de</a><mailto:<a href="mailto:magdalena.schacherl@charite.de" target="_blank">magdalena.schacherl@charite.de</a>><br>
<br>
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<br>
<br>
Von: 3dem <<a href="mailto:3dem-bounces@ncmir.ucsd.edu" target="_blank">3dem-bounces@ncmir.ucsd.edu</a>> im Auftrag von Umar Farook <<a href="mailto:umarfarook12@gmail.com" target="_blank">umarfarook12@gmail.com</a>><br>
Datum: Donnerstag, 4. Juni 2020 um 21:25<br>
An: "<a href="mailto:3dem@ncmir.ucsd.edu" target="_blank">3dem@ncmir.ucsd.edu</a>" <<a href="mailto:3dem@ncmir.ucsd.edu" target="_blank">3dem@ncmir.ucsd.edu</a>><br>
Betreff: [ext] [3dem] Hexamer dissociate while freezing Cryo-EM grids<br>
<br>
Dear All,<br>
<br>
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.<br>
<br>
Best Regards,<br>
Umar<br>
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End of 3dem Digest, Vol 154, Issue 4<br>
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</blockquote></div><br clear="all"><br>-- <br><div dir="ltr" class="gmail_signature"><div dir="ltr"><div><div dir="ltr"><div><div dir="ltr"><div><div dir="ltr"><div><div dir="ltr"><div><div dir="ltr"><div><div dir="ltr"><div><div dir="ltr"><div><div dir="ltr"><div><div dir="ltr"><div><div dir="ltr"><div><div dir="ltr"><div><div dir="ltr"><div style="background-color:rgb(255,255,255)" dir="ltr"><div dir="ltr" style="font-size:12.8px"><span style="font-size:12.8px">________________________________</span><font size="2"><br></font></div><div dir="ltr"><font size="2"><span style="background-color:rgb(255,255,255)">Elad Binshtein, Ph.D.</span></font></div><div dir="ltr"><span><font size="2">Cryo-EM specialist - Senior Scientist</font></span><font size="2"><br style="background-color:rgb(255,255,255)">Crowe lab - Vanderbilt Vaccine Center (vvc)<br>MRB4 Room 11475<br style="background-color:rgb(255,255,255)"><span style="background-color:rgb(255,255,255)">Vanderbilt University</span> Medical Center<br style="background-color:rgb(255,255,255)"><span style="background-color:rgb(255,255,255)">Nashville, TN</span><br><span style="background-color:rgb(255,255,255)">Mobile: <span><span><span><span>+1-615-481-4408</span><span dir="ltr"><span dir="ltr"><span><span></span></span></span></span></span></span></span></span><span style="background-color:rgb(255,255,0)"><span></span></span></font></div><div dir="ltr"><font size="2"><a href="https://urldefense.com/v3/__https://www.linkedin.com/in/elad-binshtein-cryoem/__;!!Mih3wA!RNO6z6ZYlKlrrQZyPOy8MgR4qlu01fuBMEARTwpoQu0efYGmufCWbmcaoMJ1arz9-w$" target="_blank">https://www.linkedin.com/in/elad-binshtein-cryoem/</a></font></div><font style="background-color:rgb(255,255,0)" size="2"><span style="background-color:rgb(255,255,255)">E-Mail: </span><a style="background-color:rgb(255,255,255)" href="mailto:eladbi@gmail.com" target="_blank">elad.binshtein@vanderbilt.edu</a><br></font></div><div style="background-color:rgb(255,255,255)" dir="ltr"><span><div dir="ltr"><font size="2">twitter @EladBinshtein</font></div></span><font style="background-color:rgb(255,255,0)" size="2"></font></div><div><span style="font-size:12.8px">________________________________</span><br></div></div></div></div></div></div></div></div></div></div></div></div></div></div></div></div></div></div></div></div></div></div></div></div></div></div></div>