Arimura Lab

Arimura Lab

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Step 3: MagIC-cryo-EM Beads Quality Assessment

Gel code

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).

Pull down test

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

MagIC-cryo-EM*

This is the test experiment to.

* For the nucleosome, 1000~2000 nucleosomes/bead is an ideal ratio. This may vary depending on your target.

Step 1: To remove too small beads with the same buffer for the sample

  1. Mix 10 µL of 3HB-60nm-SAH-GFPe beads [1 fmol], 100 µL 17% sucrose gradient buffer with 0.01% Tween20
  2. Spin 16000g 20 min 4ºC, collect the tube on the handmade magnetic rack
  3. Remove supernatant from the tube on the handmade magnetic rack

Step 2: to remove aggregated protein

  1. Transfer the sample (a fraction from a sucrose gradient containing 1~2 pmol* target complex) to a new tube (* For the nucleosome, 1000~2000 nucleosomes/bead is an ideal ratio. This may vary depending on your target.)
  2. Add Tween20 to 0.01%
  3. Spin at 16000g 20 min 4ºC
  4. Take supernatant

Step 3: Incubation

  1. Add the supernatant of Step 1 to the washed beads of Step 2
  2. Incubate (Leave 15 hrs in the cold room) overnight

Step 4: Wash

For the purified protein, this step may not be needed.

  1. Spin at 16000g 20 min 4ºC
  2. Remove supernatant
  3. Add 200 µL of EM cryoprotectant buffer with 0.01% Tween
  4. Pipette well
  5. Spin at 16000g 20 min 4ºC
  6. Remove supernatant
  7. Add 200 µL of EM cryoprotectant buffer with 0.01% Tween
  8. Pipette well
  9. Spin at 16000g 20 min 4ºC
  10. Remove supernatant
  11. Add 200 µL of EM cryoprotectant buffer with 0.01% Tween
  12. Pipette well
  13. Spin at 16000g 20 min 4ºC
  14. Remove supernatant
  15. Add 80 µL of EM cryoprotectant buffer with 0.001% Tween
  16. Pipette well
  17. Freeze grids (incubate 5 min on magnets)

Step 5: Grid freezing

  1. Plasma clean graphene grid (O₂ + H₂, 10 sec)
  2. Pick one grid with non-magnetic tweezers and set it in a magnetic humidity chamber
  3. Apply 4 µL of sample
  4. Incubate 5 min
  5. Freeze grid with Vitrobot
  6. (Skip sample application, 2-second blotting time at room temperature under 100% humidity)
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