1. Absolute Mag streptavidin nanomagnetic beads (CD Bioparticles: WHM-X047)
This magnetic bead does not cluster and stable under the electron microscope. If you want to try the magnetic beads from other vender, make sure that your bead is stable during the data collection with electron microscope.
2. Cooling centrifuge with swing-bucket rotors (e.g. Eppendorf S24-11-AT, Beckman SX241.5)
Since the beads are so small, magnetic bead pellet is invisible for human eyes. With swing-bucket rotors, even if you don’t see the pellet, you know the pellet is always at the bottom of the tube.
3. A Standard bench-top centrifuge
Our centrifuge, which achieves 2000 xg, is used to remove the aggregated beads.
4. Magnetic stand
Useful for transfer tube from centrifuge to your bench. The same magnet can be used for incubating grids in humidity chamber.
We use neodymium magnets and cut out the top part of the clear centrifuge tube, and attach it with clear tape.
5. Humidity chamber
We use a big plastic box (actually, a plastic drawer) put in a layer of paper towels on the inside to maintain the humidity and then, cover the front opening with plastic film.
6. Incubation stands for a single set of tweezers.
[link for the 3D printer file]
Incubation stands for four sets of tweezers.
[link for the 3D printer file]
7. Non-magnetic vitrobot tweezers
Important to note is that standard vitrobot tweezers are magnetic and cannot be incubated on magnets. It takes considerable strength to manage to keep the standard vitrobot tweezers from sticking to the magnet for five minutes. Therefore, non-magnetic vitrobot tweezers are recommended.
Non-magnetic Vitrobot Tweezers
8. Graphene-coated gold grids
We use homemade graphene-coated grids using Quantifoil Gold R 1.2/1.3 300 mesh grids. Graphene helps beads to stay on the grid holes during freezing on the vitrobot (if there is no graphene, you may find most of the beads on the carbon support film). If your particle has a good contrast on a micrograph, continuous thin carbon grids may also work fine.
Using gold grids is essential as copper grids are weakly magnetized by the magnetic incubation, which causes vibration during data collection.
9. Buffers
For general purposes, these are the standard buffers:
Wash buffer: 10 mM HEPES-KOH (pH 7.4), 140 mM NaCl, 3 mM KCl, 10 μg/ml leupeptin, 10 μg/ml pepstatin, 10 μg/ml chymostatin, 0.01 % Tween 20
EM buffer: 10 mM HEPES-KOH (pH 7.4), 30 mM KCl, 1 μg/ml leupeptin, 1 μg/ml pepstatin, 1 μg/ml chymostatin, 0.001 % Tween 20
0.01% Tween 20 is essential during washing to avoid the beads’ absorption by the tube and tip side wall. Other detergents may also work fine.
10. Plasmids
Plasmids for expressing the MagIC-cryo-EM proteins are available on Addgene
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Acknowledgememt
This page was designed by Antonio Latimore in the Communications & Marketing department at the Fred Hutchinson Cancer Center and maintained by Katrina Akioka and other members in the Arimura lab at the Fred Hutchinson Cancer Center.
The Original paper of MagIC-cryo-EM, has been published (Arimura Y, Konishi HA, Funabiki H. Elife. 2025). The MagIC-cryo-EM method was developed in the Funabiki lab at the Rockefeller University under support from a National Institutes of Health grant (R35GM132111) awarded to H.F., a Japan Society for the Promotion of Science Overseas Research Fellowship awarded to H.A.K., and the Osamu Hayaishi Memorial Scholarship for Study Abroad awarded to Y.A. This research was also supported by the Stavros Niarchos Foundation (SNF) as part of its grant to the SNF Institute for Global Infectious Disease Research at The Rockefeller University.
We are grateful to Mark Ebrahim, Johanna Sotiris, and Honkit Ng for their technical advice and assistance with Cryo-EM. We also thank Genzhe Lu and Daniil Tagaev for their contributions to optimizing MagIC-cryo-EM. We extend our thanks to Seth Darst, Elizabeth Campbell, Thomas Huber, Michael Rout, Peter Fridy, Christopher Caffalette, Trevor Van Eeuwen, Hiro Furukawa, Sue Biggins, Daniel Barrero, and Mengqiu Jiang for their valuable consultations on the project.
The test data were collected at the Arimura Lab and the Electron Microscopy Facility at the Fred Hutchinson Cancer Center. We are grateful to Melody Campbell, Theo Humphreys, and Anvesh Dasari for establishing and operating the Cryo-EM equipment.