If you’re struggling with poor Graphene Oxide (GO) coverage on cryo-EM grids, this simple protocol can help you achieve nearly 100% coverage. I use an adapted version of Dr. Thomas G. Martin’s protocol, originally shared here on Figshare.

Step-by-Step Protocol

1. Prepare Pyrene Stock Solution

Make a 5 mM pyrene stock by dissolving 20 mg of pyrene into 20 mL of methanol.
Store at room temperature in the dark.

2. Glow Discharge the Grids

Use a glow discharger to treat Quantifoil or C-Flat grids. This improves hydrophilicity and helps both pyrene and GO adhere more uniformly to the surface.

3. Pyrene Coating

Apply 3 μL of 0.5 mM pyrene solution to the carbon side of each grid.
Let it incubate for 1 minute at RT.

4. Graphene Oxide Application

Dilute 2 mg/mL GO (Sigma 763705) to 0.2 mg/mL in deionized water.
Apply 3 μL of the diluted GO solution to the grid.
Incubate for 2 minutes.
Tip: Longer incubation times often lead to better coverage.

5. Water Rinsing on Parafilm

Prepare three drops of deionized water on a piece of parafilm:

• Blot the front of the grid by gently touching it to the first two drops.
• Blot the back of the grid with the third drop to allow GO adhere to the grid.

6. Air Dry and Store

Allow the grid to air dry completely.
Once dry, store the GO-coated grids in a grid box.
They can be kept for years without degradation if stored in a clean, dry environment.

Final Notes

This protocol consistently yields graphene oxide grids with excellent coverage. Adjusting the GO concentration and incubation time can fine-tune results for your specific application.

Happy grid-making!