1. Absolute Mag streptavidin nanomagnetic beads (CD Bioparticles: WHM-X047)

This magnetic bead does not cluster and stable under the electron microscope. If you want to try the magnetic beads from other vender, make sure that your bead is stable during the data collection with electron microscope.

2. Cooling centrifuge with swing-bucket rotors (e.g. Eppendorf S24-11-AT, Beckman SX241.5)

Since the beads are so small, magnetic bead pellet is invisible for human eyes. With swing-bucket rotors, even if you don’t see the pellet, you know the pellet is always at the bottom of the tube.

3. A Standard bench-top centrifuge

Our centrifuge, which achieves 2000 xg, is used to remove the aggregated beads.

4. Magnetic stand

Useful for transfer tube from centrifuge to your bench. The same magnet can be used for incubating grids in humidity chamber.

We use neodymium magnets and cut out the top part of the clear centrifuge tube, and attach it with clear tape. 

5. Humidity chamber

We use a big plastic box (actually, a plastic drawer)  put in a layer of paper towels on the inside to maintain the humidity and then, cover the front opening with plastic film.

6. Incubation stands for a single set of tweezers.

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Incubation stands for four sets of tweezers.

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7.  Non-magnetic vitrobot tweezers

Important to note is that standard vitrobot tweezers are magnetic and cannot be incubated on magnets. It takes considerable strength to manage to keep the standard vitrobot tweezers from sticking to the magnet for five minutes. Therefore, non-magnetic vitrobot tweezers are recommended.

Non-magnetic Vitrobot Tweezers

8. Graphene-coated gold grids

We use homemade graphene-coated grids using Quantifoil Gold R 1.2/1.3 300 mesh grids. Graphene helps beads to stay on the grid holes during freezing on the vitrobot (if there is no graphene, you may find most of the beads on the carbon support film). If your particle has a good contrast on a micrograph, continuous thin carbon grids may also work fine.

Using gold grids is essential as copper grids are weakly magnetized by the magnetic incubation, which causes vibration during data collection.

9. Buffers

For general purposes, these are the standard buffers:

Wash buffer: 10 mM HEPES-KOH (pH 7.4), 140 mM NaCl, 3 mM KCl, 10 μg/ml leupeptin, 10 μg/ml pepstatin, 10 μg/ml chymostatin, 0.01 % Tween 20

EM buffer: 10 mM HEPES-KOH (pH 7.4), 30 mM KCl, 1 μg/ml leupeptin, 1 μg/ml pepstatin, 1 μg/ml chymostatin, 0.001 % Tween 20

0.01% Tween 20 is essential during washing to avoid the beads’ absorption by the tube and tip side wall. Other detergents may also work fine.

10. Plasmids

Plasmids for expressing the MagIC-cryo-EM proteins are available on Addgene

Plasmids for Expressing the MagIC-cryo-EM Proteins

Spacer protein purification

Because the MilliQ at the Fred Hutch is under oxidizing conditions, the protocol described in the afore mentioned paper, established at Rockefeller University, did not work at Fred Hutch.  However, using a low concentration of TECP resolved the issue.

Day 1

1. Express the spacer protein in Rosetta (DE3)
2. Resuspend E.coli pellet with sonication buffer (1x PBS, 400 mM Additional NaCl, 5 % glycerol, 5 mM 2-me, 1 mM PMSF, 30 mM imidazole, 1x protease inhibitor)
3. Sonication
4. Remove insoluble fraction with centrifugation at 30000rpm, 30min, 4ºC
5. Filter supernatant with 0.22µm syringe filter
6. Load sample to His-Trap HP 5ml column using a peristaltic pump
7. Wash column with 50 ml wash buffer 1 (1 x PBS, 800 mM Additional NaCl, 5 % Glycerol, 30 mM imidazole, 2 mM TCEP, and 1 mM PMSF)
8. Wash column with 50 ml wash buffer 2 (1 x PBS, 5 % Glycerol, 30 mM imidazole, 2 mM TCEP)
9. Connect on AKTA Pump A: 1 x PBS, 5 % Glycerol, 30 mM imidazole, 2 mM TCEP Pump B: 1 x PBS, 5 % Glycerol, 500 mM imidazole, 2 mM TCEP
10. Elute with B10% (5CV) => B40% (5CV) => B100% (7CV)
11. Run SDS-PAGE (Peak in B40% may contain spacer protein)
12. Collect fractions containing the spacer protein
13. Add SENP1 protease
14. Dialyze to 500 ml Capto HiResQ wash buffer (20mM KH2PO4 / K2HPO4 (pH8), 5% glycerol, 2 mM TCEP), 4ºC, o/n

Day 2

1. Apply the sample to His-Trap HP 5ml column and collect the flow-through
2. Apply the flow-through to Capto HiResQ
3. Elute with linear gradient
   • Pump A: 20 mM KH2PO4-K2HPO4 (pH 8), 5 % Glycerol, 2 mM TCEP
   • Pump B: 1x PBS, 1 M Additional NaCl, 5 % glycerol, 2 mM TCEP
4.  SDS-PAGE (Peak in B10-20% may contain spacer protein)
5. Collect fractions containing the spacer protein
6. Apply the sample to Superdex S200 16/600 (x1 PBS + 5% gltcerol + 1 mM TCEP)
7. SDS-PAGE (Peak in 50-60ml elution volume may contain 60 nm SAH spacer protein)
8. Collect fractions containing the spacer protein
9. Concentrate the protein using Amicon 10K to 1ml
10. Estimate the concentration using nanodrop (The extinction coefficient of 60 nm SAH is: 11460)
11. Mix Meleimide-biotin with proteins (50-fold molar excess Meleimide-biotin)
12. On ice, o/n

Day 3

1. Dialyze sample to Quench buffer (1x PBS, 5 % Glycerol, 2 mM 2-me)
2. Maintain in Cold room overnight

Day 4

1. Apply sample to Superdex S200 16/600 (1x PBS, 5% glycerol) *inadequate modification on 60nm SAH disrupts the single alpha helix and changes the Superdex profile from the day 2’s result
2. Concentrate the protein using Amicon 10K to 1ml
3. Estimate the concentration using a nanodrop
4. Add equal volume of storage buffer (1x PBS, 90% glycerol)
5. Store at -20ºC

View the MagIC-cryo-EM Frequently Asked Questions