[3dem] 3dem Digest, Vol 154, Issue 4

Elad Binshtein elad.binshtein at vanderbilt.edu
Thu Jun 4 13:10:49 PDT 2020 

Hi Umar,

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.

Best,

On Thu, Jun 4, 2020 at 2:57 PM <3dem-request at ncmir.ucsd.edu> wrote:

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> Today's Topics:
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>    1. Hexamer dissociate while freezing Cryo-EM grids (Umar Farook)
>    2. Re: Hexamer dissociate while freezing Cryo-EM grids (Jay Rai)
>    3. Re: [ext]  Hexamer dissociate while freezing Cryo-EM grids
>       (Schacherl, Magdalena)
>
>
> ----------------------------------------------------------------------
>
> Message: 1
> Date: Thu, 4 Jun 2020 22:24:44 +0300
> From: Umar Farook <umarfarook12 at gmail.com>
> To: 3dem at ncmir.ucsd.edu
> Subject: [3dem] Hexamer dissociate while freezing Cryo-EM grids
> Message-ID:
>         <CAFGvJnbAJwN6N-SiavXc1uH4Dh=
> ESCP7C7oWC9ZXdscj9et_pA at mail.gmail.com>
> Content-Type: text/plain; charset="utf-8"
>
> Dear All,
>
> 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.
>
> Best Regards,
> Umar
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> >
>
> ------------------------------
>
> Message: 2
> Date: Thu, 4 Jun 2020 19:26:47 +0000
> From: Jay Rai <jrai at fsu.edu>
> To: Umar Farook <umarfarook12 at gmail.com>, "3dem at ncmir.ucsd.edu"
>         <3dem at ncmir.ucsd.edu>
> Subject: Re: [3dem] Hexamer dissociate while freezing Cryo-EM grids
> Message-ID:
>         <
> DM6PR02MB5276D19D53787E0F8196B1B1D0890 at DM6PR02MB5276.namprd02.prod.outlook.com
> >
>
> Content-Type: text/plain; charset="us-ascii"
>
> Your sample might destroy by air-water interference.
> Remedy- try using detergent.
> Jay
>
> With kind regards,
> Jay
> ________________________________
> From: 3dem <3dem-bounces at ncmir.ucsd.edu> on behalf of Umar Farook <
> umarfarook12 at gmail.com>
> Sent: Thursday, June 4, 2020 3:24:44 PM
> To: 3dem at ncmir.ucsd.edu <3dem at ncmir.ucsd.edu>
> Subject: [3dem] Hexamer dissociate while freezing Cryo-EM grids
>
> Dear All,
>
> 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.
>
> Best Regards,
> Umar
> -------------- next part --------------
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>
> ------------------------------
>
> Message: 3
> Date: Thu, 4 Jun 2020 19:57:14 +0000
> From: "Schacherl, Magdalena" <magdalena.schacherl at charite.de>
> To: Umar Farook <umarfarook12 at gmail.com>
> Cc: "3dem at ncmir.ucsd.edu" <3dem at ncmir.ucsd.edu>
> Subject: Re: [3dem] [ext]  Hexamer dissociate while freezing Cryo-EM
>         grids
> Message-ID: <FD1B8CF0-DDDB-4F85-AC07-0C44304C02CF at charite.de>
> Content-Type: text/plain; charset="utf-8"
>
> Hi Umar,
>
> 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.
>
> 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.
>
> 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).
>
> Best regards,
>
> Magdalena
>
>
>
> ------------------------------------------------------------------------------------------------
> Magdalena Schacherl, PhD
> Group Leader Structural Enzymology
> Charit? - Universit?tsmedizin Berlin
> Institute of Medical Physics and Biophysics  | Charit?platz 1 | 10117
> Berlin
> Internal address: Virchowweg 6
> Room 03.322, level 3
> T: +49 30 450 524196 | F: +49 30 450 524952
> magdalena.schacherl at charite.de<mailto:magdalena.schacherl at charite.de>
>
> Website Schacherl lab<
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> >
>
>
>
>
> Von: 3dem <3dem-bounces at ncmir.ucsd.edu> im Auftrag von Umar Farook <
> umarfarook12 at gmail.com>
> Datum: Donnerstag, 4. Juni 2020 um 21:25
> An: "3dem at ncmir.ucsd.edu" <3dem at ncmir.ucsd.edu>
> Betreff: [ext] [3dem] Hexamer dissociate while freezing Cryo-EM grids
>
> Dear All,
>
> 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.
>
> Best Regards,
> Umar
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> ------------------------------
>
> End of 3dem Digest, Vol 154, Issue 4
> ************************************
>


-- 
________________________________
Elad Binshtein, Ph.D.
Cryo-EM specialist - Senior Scientist
Crowe lab - Vanderbilt Vaccine Center (vvc)
MRB4 Room 11475
Vanderbilt University Medical Center
Nashville, TN
Mobile: +1-615-481-4408
https://urldefense.com/v3/__https://www.linkedin.com/in/elad-binshtein-cryoem/__;!!Mih3wA!RNO6z6ZYlKlrrQZyPOy8MgR4qlu01fuBMEARTwpoQu0efYGmufCWbmcaoMJ1arz9-w$ 
E-Mail: elad.binshtein at vanderbilt.edu <eladbi at gmail.com>
twitter @EladBinshtein
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