Abstract
Blotting times for conventional cryoEM specimen preparation complicate time-resolved studies and lead to some specimens adopting preferred orientations or denaturing at the air–water interface. Here, it is shown that solution sprayed onto one side of a holey cryoEM grid can be wicked through the grid by a glass-fiber filter held against the opposite side, often called the `back', of the grid, producing a film suitable for vitrification. This process can be completed in tens of milliseconds. Ultrasonic specimen application and through-grid wicking were combined in a high-speed specimen-preparation device that was named `Back-it-up' or BIU. The high liquid-absorption capacity of the glass fiber compared with self-wicking grids makes the method relatively insensitive to the amount of sample applied. Consequently, through-grid wicking produces large areas of ice that are suitable for cryoEM for both soluble and detergent-solubilized protein complexes. The speed of the device increases the number of views for a specimen that suffers from preferred orientations.
Funder
Natural Sciences and Engineering Research Council of Canada
Canadian Institutes of Health Research, Institute of Genetics
Canada Research Chairs
Publisher
International Union of Crystallography (IUCr)
Cited by
32 articles.
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