The Peel-Blot Technique: A Cryo-EM Sample Preparation Method to Separate Single Layers From Multi-Layered or Concentrated Biological Samples

The peel-blot technique allows for the separation of multi-layered and concentrated biological cryo-EM samples into single layers to reduce thickness, increase concentration, and facilitate image processing. Sample concentrations can be increased prior to grid preparation, and the peel-blot can be adjusted to result in a dense distribution of single-layered specimens for highly efficient data collection. To begin, stack two pieces of 20-25 micrometer pore size filter paper and place a long paraffin film adjacent to it with the paper facing up.

Place a piece of submicron filter paper on top of two additional pieces of filter paper. Position the anti-capillary forceps with the bent leg facing up and the straight leg facing down and select a 600 mesh grid with the smooth or shiny side up. Place the forceps on a Petri dish or other raised surface to avoid contact of the clean grid with other items.

Remove the paper from the paraffin film and pull the sides to create a small rim. Pipette two 150 microliter drops so 4%trehalose solution at around one to two centimeters from one short side of the paraffin film. The two drops should be around one centimeter apart.

Next, on a separate bench or on the floor, pour liquid nitrogen into a small polystyrene foam container and place a small cryo-EM grid container in the liquid nitrogen. Cover the polystyrene foam container with lint-free wipes. To float the carbon film from the mica, use Dumont 5 Forceps and touch one side of the mica at a slight angle downwards on one of the drops of trehalose solution.

Move the mica downwards to let the water tension lift off the carbon film slowly. Release the mica once the carbon film floats on the surface of the droplet. Visually inspect the carbon film to avoid using carbon with damage in the form of fractures.

Insert the grid held by the anti-capillary forceps at a 20-30 degree angle into a trehalose drop at a position adjacent to, and then center the grid under the carbon film. Pick up the carbon film, keep the grid in the same orientation and slowly lower the grid onto the surface of the second drop of trehalose without breaking the drop’s surface tension. This will remove unnecessary carbon film that may have wrapped around the rim of the grid to the rough side.

Rotate the anti-capillary forceps and place them on a Petri dish with the carbon side of the grid facing down. Pipette 1.3 microliters of the sample into the slight trehalose meniscus on top of the grid. Then, pipette the solution approximately 8-10 times to mix and distribute the trehalose and the sample on both sides of the grid.

Pipette one or more 1.7 microliter drops of trehalose solution onto the paraffin film while incubating the sample trehalose solution on the grid for one minute. Rotate the grid back into its original position with the carbon film facing up and press the grid onto the submicron filter paper using the anti-capillary forceps. Once the solution has been absorbed by the filter paper and the carbon film is lying flat, wait for three seconds before lifting the grid and moving it vertically onto the 1.7 microliter drop of trehalose solution.

These steps can be repeated between one to three reiterations or more, depending on the sample. Blot the grid on the two pieces of filter paper and then lift the grid from the filter paper. After approximately 13 seconds, depending on humidity and sample buffer, plunge the grid into the liquid nitrogen in the small styrofoam container and place it in the cryo-EM grid container or transfer it directly for cryo-EM screening or data collection.

A 2D crystal sample of human leukotriene C4 synthase subjected to the peel-blot method is shown here. The region to the left of the arrow was largely reduced to single-layered 2D crystals in a contact region between the carbon film and the grid bar that was peel-blotted and then shifted. The region to the right of the arrow was not affected by the peel-blot.

The lower half of the image represents the regions with large, mostly single-layered phospho lipid bilayers resulting from the application of the peel-blot. The upper half was not in a contact zone between the grid bar and the carbon film, and thus contained complete liposomes with two phospholipid bilayers. The grid square of a 400 mesh grid shows the footprint of the grid bar and the resulting peel-blotted regions as well as the regions that were not in contact with a grid bar or subjected to fewer peel-blot iterations.

These images showed the blotting of an EM grid prepared by the back injection on a submicron filter paper using very thin carbon film. The images are single frames at initial contact with the membrane, 1, 000 milliseconds after contact, and 2, 000 milliseconds after contact. The arrow indicates grid squares where capillary pressure ruptured the carbon film.

Avoiding fractures in the carbon film and combining submicron filter paper with a 1.7 microliter trehalose drop to suction and then separate the layers are key steps. The peel-blotted samples are used for high resolution cryo-EM data collection, followed by image processing for the structure determination. The peel-blot allows for structured determination of single-layered protein 2D crystals which were previously too thick for cryo-EM.

Furthermore, it can concentrate various samples into single layers on carbon film.