Day 1
1. Transform BL21(DE3) with SPYtag-Histag-Avidin, Non tagged Avidin
2. LB (Carb) plate, 37 ºC O/N
Day 2
3. Inoculate E.Coli to 1L x2
4. Add 1 mM IPTG
5. 37ºC O/N (No need to reduce temperature)
Day 3
6. SDS-PAGE
7. Harvest
8. Add 100ml sonication buffer (50mM Tris-HCl, 1mM EDTA)
9. Sonication (2sec on/5sec off, 100%, total 10min on time)
10. Spin at 20,000rpm, 4ºC, 30min
11. Remove sup
12. Add 50 ml sonication buffer
13. Sonication
14. Spin at 20,000rpm, 4ºC, 30min
15. Remove sup
16. Resuspend ppt with 8ml 6M Gdn-HCl (pH 1.5)
17. Dialyze to 200ml 6M Gdn-HCl (pH 1.5) O/N, (Pore size:10K)
Day 4
18. Spin 20,000rpm, 4ºC, 20min
19. Collect sup
20. Nanodrop
(Non-tag: Ext. coefficient 41940, Abs 0.1% (=1 g/l) 3.129, His-SPY: Ext. coefficient 44920, Abs 0.1% (=1 g/l) 2.783)
21. Mix Non-tag streptavidin and SPY-His- streptavidin at a molar ratio of 4:1
22. Drop refolding to 250ml PBS (on stirrer) in a cold room
23. stir at 4ºC, 3 hrs
24. Spin at 20,000rpm, 4ºC, 30min
25. Collect sup
26. Slowly add 62.7g of solid ammonium sulfate in a cold room
27. Stair at 4ºC, 3 hrs
28. Spin at 20,000rpm, 4ºC, 30min
29. Collect sup
30. Slowly add 59 g of solid ammonium sulfate in cold room
31. Stair at 4ºC, O/N
Day 5
32. Spin at 20,000rpm, 4ºC, 30min x2
33. Resuspend ppt with 50 ml of PBS with 2.2M ammonium sulfate
34. Spin at 20,000rpm, 4ºC, 30min
35. Resuspend ppt with 5 ml (remove ppt)
36. Hi-load S75 16/600 (use PBS)
37. SDS-PAGE (with or without heat)
38. Collect fractions
39. Apply samples to the HisTrap HP 5ml column on AKTA and elute with liner gradient* (*important)
(Buffer A: PBS + 10 mM imidazole, Buffer B: PBS + 500 mM imidazole)
→ This will elute proteins as multiple peaks, depending on how many SPY-His-streptavidin are contained in the streptavidin tetramer
40. SDS-PAGE (with and without pre-heating samples)
=> SDS-PAGE without pre-heating samples is useful to distinguish how many SPY-His-streptavidin are contained in the streptavidin tetramer
41. Collect Fractions with mono SPY-tag-streptavidin tetramer
42. Dialyze to 500ml PBS (12-14kDa cut-off) O/N
43. Dialyze to fresh 500ml PBS (12-14kDa cut-off) 4hrs
44. Nanodrop (Ext. coefficient of mono-SPY-His-SPYtag avidin tetramer: 170,740)
45. Amicon 10K
46. Nanodrop
47. Add an equal volume of PBS with 90% glycerol.
48. Store at -20ºC
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.