Part of this work was supported by NIH grant HL090630 (ISK).

1. Preparation steps for the peel-blot technique
NOTE: Preparation requires for the following items to be placed adjacent to each other.
<ol>
	<li>Stack two pieces of 20-25 &#181;m pore size filter paper (see Table of Materials).</li>
	<li>Place an ~8 cm x 10 cm long paraffin film adjacent to the filter paper with the paper facing up.</li>
	<li>Place a piece of submicron filter paper on top of two additional pieces of #4 filter paper (Figure 1-1).</li>
	<li>Position the anti-capillary forceps with the bent leg facing up and the straight leg facing down. Then, select a 600-mesh grid with the smooth/shiny side up. Place the forceps on a Petri dish or other raised surface to avoid contact of the clean grid with other items.</li>
	<li>Remove the paper from the paraffin film and pull the sides (~5 mm) to create a small rim. Pipette two 150 &#181;L drops of 4% trehalose solution ~1-2 cm from one short side of the paraffin film and ~1 cm apart on the paraffin film.</li>
	<li>On a separate bench or on the floor, pour liquid nitrogen in a small polystyrene foam container (see Table of Materials). 
	CAUTION: Receive training for proper handling using gloves and a face shield in order to avoid frost bite.</li>
	<li>Place a small cryo-EM grid container in the liquid nitrogen and cover the polystyrene foam container with lint-free wipes.</li>
</ol>
2. Placing the carbon film onto the grid
<ol>
	<li>Use Dumont #5 forceps (see Table of Materials) to float of the carbon film from the mica by touching one side of the mica at a slight angle downwards (~20&#176;-40&#176;) on one of the drops of trehalose solution and slowly let the water tension lift off the carbon film by moving the mica downwards. Release the mica once the carbon film floats on the surface of the droplet.</li>
	<li>Visually inspect the carbon film to avoid using carbon with damage in the form of fractures.</li>
	<li>Insert the grid held by the anti-capillary forceps at an angle (~20&#176;-30&#176;) into a trehalose drop at a position adjacent to, and then centered under the carbon film. Pick up the carbon film (Figure 1-2).</li>
	<li>Keep the grid in the same orientation and slowly lower it onto the surface of the second drop of trehalose without breaking the surface tension of the drop to remove unnecessary carbon film that may have wrapped around the rim of the grid to the rough side.</li>
</ol>
3. Separation of multi-layered 2D crystals into single layers
<ol>
	<li>Rotate the anti-capillary forceps (see Table of Materials) and place them on a Petri dish with the carbon side of the grid facing down (Figure 1-3).</li>
	<li>Pipette 1.3 &#181;L of the sample into the slight trehalose meniscus on top of the grid. Pipette the solution ~8-10 times to mix and distribute the trehalose and sample on both sides of the grid.</li>
	<li>While incubating the solution for 1 min, pipette one or more 1.7 &#181;L drops of trehalose solution onto the paraffin film.</li>
	<li>Rotate the grid back into its original position with the carbon film facing up.</li>
	<li>Use the anti-capillary forceps to press the grid onto the submicron filter paper (Figure 1-4). Once the solution has been absorbed by the filter paper and the carbon film is lying flat, wait for 3s before lifting the grid and moving it vertically onto the 1.7 &#181;L drop of trehalose solution (Figure 1-5). 
	NOTE: Repeat this step multiple times for highly stacked samples, or the grid is prepared for vitrification in the next steps.</li>
	<li>Blot the grid on the two pieces of filter paper, and then lift the grid from the filter paper (Figure 1-6). After approximately 13 s, depending on humidity and sample buffer, plunge the grid into the liquid nitrogen in the small Styrofoam container and place the grid in the cryo-EM grid container or transfer it directly for cryo-EM screening or data collection. 
	NOTE: In order to rapidly optimize the protocol, negatively stain the peel-blotted grid at this step rather than plunging it into liquid nitrogen. Negatively stain the sample by applying 3 &#181;L of 1% uranyl acetate to the grid, incubate the grid for 30 s, and blot it with a torn piece of 20-25 &#181;m pore size filter paper. Grids of 2D crystals vitrified in trehalose, tannin, or glucose are routinely hand-plunged for vitrification as trehalose, tannin, and glucose prevent the formation of crystalline ice at rates used for hand-plunging2.</li>
</ol>

<ol>
	<li>Johnson, M. C., Uddin, Y. M., Neselu, K., Schmidt-Krey, I. 2D crystallography of membrane protein single-, double- and multi-layered ordered arrays. Methods in Molecular Biology. 2215, 227-245 (2021).</li>
	<li>Ku&#776;hlbrandt, W., Downing, K. H. Two-dimensional structure of plant light harvesting complex at 3.7 &#197; resolution by electron crystallography. Journal .of Molecular Biology. 207&#160;(4), 823-828 (1989).</li>
	<li>Nannenga, B. L., Gonen, T. MicroED: a versatile cryoEM method for structure determination. Emerging Topics in Life Sciences. 2&#160;(1), 1-8 (2018).</li>
	<li>Martynowycz, M. W., Zhao, W., Hattne, J., Jensen, G. J., Gonen, T. Collection of continuous rotation microED data from ion beam-milled crystals of any size. Structure. 27&#160;(3), 545-548 (2019).</li>
	<li>Gonen, T. et al. Lipid-protein interactions in double-layered two-dimensional AQP0 crystals. Nature. 438&#160;(7068), 633-638(2005).</li>
	<li>Gonen, T. The collection of high-resolution electron diffraction data. Methods in Molecular Biology. 955, 153-169 (2013).</li>
	<li>Wisedchaisri, G., Gonen T. Phasing electron diffraction data by molecular replacement: strategy for structure determination and refinement. Methods in Molecular Biology. 955, 243-272 (2013).</li>
	<li>Gipson B., Zeng X., Zhang Z. Y., Stahlberg, H. 2dx-user-friendly image processing for 2D crystals. Journal of Structural Biology. 157&#160;(1), 64-72 (2007).</li>
	<li>Biyani, N. et al. Focus: The interface between data collection and data processing in cryo-EM. Journal of Structural Biology. 198&#160;(2), 124-133 (2017).</li>
	<li>Righetto, R., Stahlberg, H.Single particle analysis for high-resolution 2D electron crystallography. Methods in Molecular Biology. 2215, 267-284 (2021).</li>
</ol>

The authors do not have competing financial interests or other conflicts of interest.

The peel-blot is a powerful method to overcome stacking and thickness of multi-layered 2D crystals and similar samples for cryo-EM data collection and image processing. A decision on the use of the peel-blot will be based on biological parameters of the sample and will also require assessment once a 3D cryo-EM model is available. The biological importance of contacts and potential flexibility of contact regions will be particularly important in this assessment.
The number of peel-blots will in...

The peel-blot technique was developed in order to separate stacked two-dimensional crystals of membrane proteins into single layers to obtain suitably thin samples for cryo-EM 2D and 3D data collection and facilitate image processing1. The protocol is similarly suitable for other types of multilamellar samples as well as for achieving high sample concentrations of either sample type on the grid without increasing the thickness in Z.
Reduction of sample layers to one layer is achieved by applying a combination of capillary pressure and shearing forces in a protocol that substantially extends on and modifies the back-injection method2. A first blotting step results in tight sandwiching and adherence to the carbon film and a grid bar on either side of the multi-layered sample during blotting on submicron rather than the 20-25&#181;m filter paper pore size in the back-injection method. The separation, or peeling, of an individual layer occurs upon forcing the separation of the sandwiched layers once the grid is pressed vertically onto a trehalose drop, forcing the carbon film away from the grid bar (Figure 1). Repositioning of the carbon film away from the original contact point with the grid bar occurs in the next step, when the grid is once again blotted on submicron filter paper. The number of iterations of these steps can be optimized for specific samples. Furthermore, the protocol can be used to increase sample concentrations while avoiding aggregation in Z. Due to the reliance on adherence to the grid bar and carbon film as well as forceful separation, this approach may not be suitable for highly fragile molecules such as certain delicate and/or unstable proteins and protein complexes.
The peel-blot technique neither requires specialized equipment nor expensive supplies. Applicability to specific samples will, however, require careful considerations of biological parameters. The decision is easier for 2D crystals as carbon film is required to ensure flatness, yet manipulation via the peel-blot technique may affect the biological integrity of the sample or crystalline order. Suitability of the peel-blot technique to single particles is restricted to and may be tested for those that require carbon film and are stable to manipulation. Specimens with critical biological interactions between layers may not be suitable for characterization with help of the peel-blot technique due to the disruption of such interactions.
The peel-blot technique (&#34;peel-blot&#34;) is most quickly optimized by testing and screening with negative stain or unstained samples by TEM at room temperature. Once the optimal number of peel-blot iterations have been identified, the time to control thickness before vitrification can be determined.

<table><tbody><tr><td>600-mesh grids</td><td>SPI</td><td>2060-C-XA</td><td></td></tr><tr><td>Anti-capillary forceps</td><td>Ted Pella</td><td>510-5</td><td></td></tr><tr><td>Carbon to coat mica</td><td></td><td></td><td></td></tr><tr><td>Cryo-EM</td><td></td><td></td><td>Cryo-EM grids may be screened at 120 kV or 200 kV.&amp;nbsp; High-resolution data is collected at 300 kV.</td></tr><tr><td>Dumpont #5 forceps</td><td>Ted Pella</td><td>5622</td><td></td></tr><tr><td>Grid box for cryo-EM storage</td><td>Ted Pella</td><td>160-40</td><td></td></tr><tr><td>Kim Wipes</td><td></td><td></td><td></td></tr><tr><td>Liquid nitrogen</td><td></td><td></td><td></td></tr><tr><td>Mica</td><td>Ted Pella</td><td>56</td><td>The mica is carbon-coated and cut into squares that are slighlty larger than a TEM grid.&amp;nbsp; The carbon thickness may require optimization to avoid thin carbon that breaks easily upon multiple peel blots.</td></tr><tr><td>Negative stain</td><td></td><td></td><td>1-2% uranyl acetate is suitable for many samples.&amp;nbsp; Other stains such as phosphotungstic acid can be substituted.</td></tr><tr><td>Parafilm</td><td></td><td></td><td></td></tr><tr><td>Polystyrene container</td><td></td><td></td><td>Used for vitrifying the peel-blot grid. A polystyrene shipping container can be recycled for this purpose and lined with aluminium foil.</td></tr><tr><td>Submicron filter paper</td><td>MilliporeSigma</td><td>DAWP04700</td><td></td></tr><tr><td>Transmission electron microscope</td><td>JEOL</td><td>JEM-1400</td><td>Any TEM operated at an accelerating voltage of 80-120 kV will be suitable for screening of negatively stained grids.</td></tr><tr><td>Trehalose</td><td></td><td></td><td>Prepare 4% trehalose solution.</td></tr><tr><td>Whatman #4 filter paper</td><td>MilliporeSigma</td><td>WHA1004150</td><td>This corresponds to the 20-25 &amp;mu;m pore size filter paper in the protocol.</td></tr></tbody></table>

the peel-blot technique: a cryo-em sample preparation method to separate single layers from multi-layered or concentrated biological samples

The peel-blot cryo-EM grid preparation technique is a significantly modified back-injection method with the objective of achieving a reduction in layers of multi-layered samples. This removal of layers prior to plunge freezing can aid in reducing sample thickness to a level suitable for cryo-EM data collection, improving sample flatness, and facilitating image processing. The peel-blot technique allows for the separation of multilamellar membranes into single layers, of layered 2D crystals into individual crystals, and of stacked, sheet-like structures of soluble proteins to likewise be separated into single layers. The high sample thickness of these types of samples frequently poses insurmountable problems for cryo-EM data collection and cryo-EM image processing, especially when the microscope stage must be tilted for data collection. Furthermore, grids of high concentrations of any of these samples can be prepared for efficient data collection since sample concentration prior to grid preparation can be increased and the peel-blot technique adjusted to result in a dense distribution of single-layered specimen.

A successful peel-blot experiment will result in single layers of samples at often high concentrations. It is to be expected that some regions in most grid squares will still contain multiple layers, especially at fewer numbers of iterations of the peel-blot steps. The majority of grid squares, however, will contain extensive regions of single layers as observed for 2D crystals of human leukotriene C4&#160;synthase (Figure 2) and liposomes (added in step 3.2, ...

Watch this Scientific Journal Video about The Peel-Blot Technique: A Cryo-EM Sample Preparation Method to Separate Single Layers From Multi-Layered or Concentrated Biological Samples at JoVE.com

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 is a cryo-EM grid preparation method that allows for the separation of multilayered and concentrated biological samples into single layers to reduce thickness, increase sample concentration, and facilitate image processing.

The peel-blot cryo-EM grid preparation technique is a significantly modified back-injection method with the objective of achieving a reduction in layers ...

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