[3dem] Hydration failure on continuous carbon despite solvent cleaning

[Dan Richman]

Hi 3DEMers,
My lab had previous success with 2nm continuous carbon coated Quantifoils (QF R2/2 300 +2nm, or EMS cat # Q3100CR2-2NM), but hydration and particle adsorption stopped working when we got two new boxes (both are lot 201027). I tested the chloroform, acetone, isopropanol cleaning procedure from Passmore and Russo 2016, checked by negative stain and a very stable control protein (confirmed to work on a CF400-UL grid with the same glow discharge and stain procedure). On the QF+2nm with or without cleaning, particles aggregate in pooled stain patches (images attached). The failure in cryo-EM is similar, very dry squares, sometimes with central pools, but hardly any particles.

Has anyone seen this behavior with continuous carbon grids, especially the commercial coatings? Any recommendations for other cleaning techniques?

Unfortunately the glow discharge treatment has to be quite gentle otherwise the thin coating over the holes will break - we use 15 mA for 15 sec and then try to compensate with longer droplet incubation times, 20-60 seconds.

Thanks for any advice!

--
Dan Richman, PhD   (he/him/his)
Research Associate, Berger Lab
Dept of Biophysics and Biophysical Chemistry
Johns Hopkins University School of Medicine
mobile: 201-669-0967
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