Sample Preparation using a Lipid Monolayer Method for Electron Crystallographic Studies

Over the years, lipid Monolayers have been used as a supporting layer to foster the 2D crystallization of peripheral membrane proteins, as well as soluble proteins. This method can also be applied to 2D crystallization, as some integral membrane proteins. But requires more extensive empirical investigation to determine detergent and dialysis conditions to promote partitioning to the lipid monolayer.

A lipid monolayer forms at the air water interface, such that the polar head groups of the lipid remain hydrated in the aqueous phase. And the non-polar acyl tails of the lipid partition to the air water interface, which breaks the surface tension and flattens the water surface. The charge nature the distinctive chemical moieties of the lipid head groups provide the affinity for proteins in solution.

Promoting binding, and in principle 2D array formation. Any 2D crystals formed on a lipid monolayer can be readily transferred into the electron microscope on a carbon coated EM grid that is used to lift and support the crystalline array on the lipid layer. We described in this manuscript, a lipid monolayer methodology for cryogenic electron microscopy imaging.

Lipid monolayer preparation. Lipid stock preparation, prepare a 0.01 mg per ml lipid mixture in 9:1 volume to volume chloroform, methanol. Using 8.91 ml of chloroform and 0.99 ml of methanol and 0.1 mls of a 10 mg per ml lipid solution.

Teflon plate preparation. Sonicate a Teflon plate in a methanol bath for five minutes. Wash with hot water for 15 minutes and then rinse with distilled water.

Dry the Teflon plate in a desiccated for 30 minutes and keep it under vacuum until use. Formation of lipid monolayer on buffer reservoir. Estimated operating time will be one and a half hours.

Place a type one filter paper in a Petri dish and arrange the Teflon block on top of the filter paper. Fill the wells of the Teflon plate with 60 microliters of buffer. Carefully add liquid mixture on top of the buffer surface drop by drop.

Ideally one microliter per drop using a Hamilton syringe. Wet the filter paper with distilled water and keep humidity in the Petri dish. Incubate at room temperature for 60 minutes, the chloroform should evaporate and a monolayer of lipid on the surface of the buffers should be formed.

Application of an EM grid on a lipid monolayer. Estimated operating time one and a half hours. Gently place a carbon coated EM grid without glow discharging carbon side down onto the surface of each buffer reservoir.

Carefully inject proteins into the side injection well, use final protein concentrations in the well around one micromolar. Incubate 60 minutes at room temperature. Gently inject approximately 20 to 30 microliters of buffer in the site injection port to raise the grid above the surface of the Teflon block.

This allows you to pick up the grid with a pair of tweezers and lift it vertically off the droplet. Representative results. A lipid monolayer deposited on an EM grid can be visualized under the transmission electron microscope without contrast staining.

The monolayer presence can be recognized, by the contrast difference from the area without any specimen in that beam path. Areas that have lipid monolayer coverage have lower contrast than ones with no coverage. Since the electron beam through the empty holes has no scattering it shows a brighter illumination.

In conclusion, the lipid monolayer method offers an opportunity for studying protein structures due to its simplicity. It may provide an alternative approach to facilitate the process of 2D crystallization.