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

[Felipe Merino]

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 <mailto: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> 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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-- 
Felipe Merino,
Max Planck Institute for Developmental Biology
Department of Protein Evolution
Max-Planck-Ring 5
72076 Tuebingen
Phone: +49 7071 601 1351

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