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

Summary

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.

Abstract

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.

Introduction

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-i....

Protocol

1. Preparation steps for the peel-blot technique

NOTE: Preparation requires for the following items to be placed adjacent to each other.

1. Stack two pieces of 20-25 µm pore size filter paper (see Table of Materials).
2. Place an ~8 cm x 10 cm long paraffin film adjacent to the filter paper with the paper facing up.
3. Place a piece of submicron filter paper on top of two additional pieces of #4 filter paper (Fi.......

Discussion

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.......

Materials

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