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

[Schacherl, Magdalena]

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


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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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