[3dem] [ext] Hexamer dissociate while freezing Cryo-EM grids

[Umar Farook]

Thanks everyone for the quick response.

Best regards,
Umar

On Fri, 5 Jun 2020, 5:09 am Jay Rai, <jrai at fsu.edu> wrote:

> 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.
>
>
> With kind regards,
> Jay
> ------------------------------
> *From:* 3dem <3dem-bounces at ncmir.ucsd.edu> on behalf of Felipe Merino <
> felipe.merino at tuebingen.mpg.de>
> *Sent:* Thursday, June 4, 2020 4:12:28 PM
> *To:* 3dem at ncmir.ucsd.edu <3dem at ncmir.ucsd.edu>
> *Subject:* Re: [3dem] [ext] Hexamer dissociate while freezing Cryo-EM
> grids
>
>
> Hi Umar,
>
> 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.
>
> Best,
>
> Felipe
> On 04.06.20 21:57, Schacherl, Magdalena wrote:
>
> 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*
>
> Charité - Universitätsmedizin Berlin
>
> Institute of Medical Physics and Biophysics  | Charitéplatz 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
>
>
>
> Website Schacherl lab
> <https://urldefense.com/v3/__https://biophysik.charite.de/en/research/structural_enzymology_schacherl_lab/__;!!Mih3wA!RCBOFKYqP30RiDg5ED5tufy2vsc_-ByfjO5mvQAv7w-7fIIqLsbhR2y2YIoqQEZRjQ$>
>
>
>
>
>
>
>
>
>
> *Von: *3dem <3dem-bounces at ncmir.ucsd.edu> <3dem-bounces at ncmir.ucsd.edu>
> im Auftrag von Umar Farook <umarfarook12 at gmail.com>
> <umarfarook12 at gmail.com>
> *Datum: *Donnerstag, 4. Juni 2020 um 21:25
> *An: *"3dem at ncmir.ucsd.edu" <3dem at ncmir.ucsd.edu> <3dem at ncmir.ucsd.edu>
> <3dem at ncmir.ucsd.edu>
> *Betreff: *[ext] [3dem] Hexamer dissociate while freezing Cryo-EM grids
>
>
>
> Dear All,
>
>
>
> I would like to ask the community for suggestions on how to overcome the
> issue of hexamer dissociating while freezing Cryo-EM grids, the negative
> staining seems to be perfect hexamer at 0.1 mg/ml concentration. But the
> sample seems to fall apart at 1.5 mg/ml in Cryo freezing, please advise,
> thank you.
>
>
>
> Best Regards,
>
> Umar
>
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> --
> Felipe Merino,
> Max Planck Institute for Developmental Biology
> Department of Protein Evolution
> Max-Planck-Ring 5
> 72076 Tuebingen
> Phone: +49 7071 601 1351
>
> ORCID:  https://urldefense.com/v3/__https://orcid.org/0000-0003-4166-8747__;!!Mih3wA!VM_H8HIiWJkjJc7345zfKqzG4yioVhdlW_vDYJ59N2ce7tEGe-SJiWmQrDJ5Xr29VQ$  <https://urldefense.com/v3/__https://orcid.org/0000-0003-4166-8747__;!!Mih3wA!Wovm-gVQiGlBAGs2Q9dEbwbScSTOZHyMZnEfw6hf5aactNdJuD44gYtwQi4DHBc_cA$>
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