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

[Jay Rai]

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<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><mailto:3dem-bounces at ncmir.ucsd.edu> im Auftrag von Umar Farook <umarfarook12 at gmail.com><mailto:umarfarook12 at gmail.com>
Datum: Donnerstag, 4. Juni 2020 um 21:25
An: "3dem at ncmir.ucsd.edu"<mailto:3dem at ncmir.ucsd.edu> <3dem at ncmir.ucsd.edu><mailto: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
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