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Graphene Oxide (GO) Grid Preparation Protocol

This protocol describes how to prepare 1-3 layers of semi-amorphous graphene oxide (GO) on Quantifoil grids, suitable for cryo-EM. GO films demonstrate reduced inelastic scattering and lower background compared to amorphous carbon, which improves phase contrast for imaging smaller complexes. In addition, it allows working with lower protein concentration and avoid prefered orientation. This protocol ensures 40-80% coverage of GO after vitrification, with precise handling steps to minimize aggregation and variation in coverage.

Protocol based on Ban Lab protocol, ETH Zurich.

Materials and Equipment

1. Preparation of Grids

  1. Ethyl Acetate Treatment:
    • Place the grids in a glass petri dish on filter paper.
    • Add ~1 mL ethyl acetate to the dish (ensure the grids do not float).
    • Leave the grids overnight to soak.
    • If needed, dry the grids in a hood the next morning.

2. Preparation of GO Dispersion

  1. Mix the Stock Solution:

    • Invert the 2 mg/mL GO dispersion (Sigma-Aldrich #763705) 10 times to ensure homogeneity.
    • Use a 1 mL pipette tip to gently mix the stock.

  2. Dilute the Stock Solution:

    • Prepare a 0.2 mg/mL GO dispersion by diluting the stock in MilliQ water.
      • Example: For 1.5 mL of 0.2 mg/mL solution, mix 150 µL stock with 1.35 mL MilliQ.

  3. Sonication of GO Dispersion:

    • Sonicate the prepared GO solution for 4 minutes at 100% power using a water sonicator. The bath should be filled with water to the level indicated and Eppendorf tubes should be placed in a floating rack.
      • Note: Avoid longer sonication times, which can damage the GO sheets.

  4. Remove Aggregates:

    • Centrifuge the sonicated GO solution at 500 g for 1 minute.
    • Carefully transfer only the upper half of the supernatant to a new Eppendorf tube to avoid aggregates. There will be a small pellet at the bottom and dark pellet on a side. Try getting the liquid from the opposite side of the dark pellet.

3. Coating Grids with GO

  1. Prepare the Workstation:

    • Wash the tweezers and prepare a sheet of Parafilm.
    • Add 20 µL MilliQ water droplets to the Parafilm (two droplets per grid).

  2. Glow Discharge the Grids:

    • Glow discharge the cleaned grids at 15 mA for 30 seconds.
    • Caution: Avoid placing the glow-discharged side on filter paper to prevent contamination. Take each grid into the tweezers by the side.

  3. Apply GO Solution to Grids:

    • Mix the GO dispersion thoroughly by pipetting up and down before each use.
    • Add 3 µL of GO solution to each grid at 15-second intervals.
    • Incubate the grids for 4 minutes to allow proper coating.

  4. Wash the Grids:

    • Blot the GO solution off the grid using the flat part of filter paper.
    • Immediately pick up the MilliQ water droplet on the Parafilm with the grid to wash it.
    • Blot the water off with filter paper.
    • Repeat this washing step for every grid.
    • Note: Make sure the grid is correctly positioned for GO deposition. Otherwise strange things happen with two CTF rings.
    • (There should be a visible GO in the blotting paper even after water washes)

  5. Drying the Grids:

    • Let the grids air-dry for at least 10 minutes.
    • Transfer the dried grids to a metal grid holder and allow them to rest for 15 minutes before further use (optional)
    • I usually use them immediately after drying (10min) and never had any issues.

4. Checking Coverage and Storage

  1. Check GO Coverage:

    • Use a TEM at ~280x magnification and low intensity with the eyepiece to search for the charging effect.
    • Aim for 60-80% coverage with 1-4 layers of GO.

  2. Storage:

    • Don’t store the grids. Use and prepare them fresh for best results.

5. Vitrification of GO Grids

  1. Vitrification Settings:
    • Use a Vitrobot or similar plunge freezer.
    • Set wait time to 15 seconds to allow the sample (3 µL) to interact with the GO surface.
    • Blot time can be adjusted based on sample thickness; start with 3 seconds. We usually use 2-4 seconds with blot force 0.
    • Humidity should be set to 100% at 4°C.
    • Drain time after blotting can be set to 0.5 seconds.

6. Notes and Troubleshooting

This protocol provides reliable preparation of graphene oxide grids with optimal thickness and coverage for cryo-EM experiments.