1. Use 25 µL of CD nanobeads-streptavidin [25fmol]
2. Add 150 µL of wash buffer
3. Add 30 µL of 6.8µM Biotin-3HB-Spycatcher3 [200pmol]
4.Leave O/N in cold room
5. Add biotin to 1 mM and mix well
6. Remove aggregation by Minispin 10 sec,
7. Transfer soluble beads to new tube
8. Spin 16000g 20min 4ºC, Remove the supernatant on the handmade magnetic rack
9. Resuspend beads with 200 µL of wash buffer
10. Spin 16000g 20min 4ºC, Remove the supernatant on the handmade magnetic rack
11. Resuspend beads with 200 µL of wash buffer
12. Add 200pmol N-tag mono SPY-avidin tetramer
13. Leave O/N in the cold room
14. Remove aggregation by Minispin 10 sec, transfer soluble beads to a new tube
15. Spin 16000g 20min 4ºC, Remove the supernatant on the handmade magnetic rack
16. Resuspend beads with 200 µL of wash buffer
17. Spin 16000g 20min 4ºC, Remove the supernatant on the handmade magnetic rack
18. Resuspend beads with 200 µL of wash buffer
19. Add 800pmol Biotin-60nm SAH-Spycatcher3
20. Leave 12 hrs in cold room
21. Add biotin to 1 mM and mix well
22. Mini-spin 10 sec, transfer soluble beads to a new tube
23. Spin 16000g 20min 4ºC, Remove the supernatant on the handmade magnetic rack
24. Resuspend the beads with 200 µL of wash buffer
25. Spin 16000g 20min 4ºC, Remove the supernatant on the handmade magnetic rack
26. Resuspend the beads with 200 µL of wash buffer
27. Add 800pmol SPY-GFP enhancer
28. Leave O/N in the cold room
29. Spin 16000g 20min 4ºC, Remove the supernatant on the handmade magnetic rack
30. Resuspend the beads with 200 µL of wash buffer
31. Spin 16000g 20min 4ºC, Remove the supernatant on the handmade magnetic rack
32. Resuspend the beads with 250 µL of wash buffer (For storage at -20ºC , resuspend beads with 50% glycerol containing wash buffer)
Continue
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.