MagIC-cryo-EM beads quality assessment (SDS-Page)
SDS-PAGE of 2~5 fmol beads can visualize the protein bands. We highly recommend running a SDS-PAGE of each step of the beads, including the streptavidin beads, before assembling the proteins (SDS-PAGE should show the bands of BSA and streptavidin).
MagIC-cryo-EM beads quality assessment (Pull down assay)
Confirming the pulldown of GFP-tagged proteins is useful for assessing the quality of the beads before using them for the cryo-EM sample, as it ensures that the sample won’t be wasted. Below is the quality check experiment done in our lab. ‘GFP-H1.8-nucleosome’ and ‘nucleosome’ can be replaced with both your positive and negative control samples. (Please contact me if you want to learn what caused the “bad beads” in the gel.)
1. Mix GFP-nanobody MagIC-cryo-EM beads with 5 pmol nucleosome or 2 pmol GFP-H1.8-nucleosome in 50 µL solution
2. Place on ice 2 hours
3. Spin tubes at 16000g, 20min, 4ºC.
4. Transfer tubes onto the handmade magnetic rack and remove the supernatant
5. Resuspend the beads with 100 µL of wash buffer
6. Spin the tubes at 16000g, 20min, 4ºC.
7. Transfer the tubes onto the handmade magnetic rack and remove the supernatant
8. Resuspend the beads with SDS sample buffer and run SDS-PAGE
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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.