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)