How to make a biological membrane in the lab

The Making of a Cell Membrane

The making of a cell membrane is a remarkable wonder of the behavior of biological molecules. It has been found that very large structures in cells are made of many, sometimes up to thousands of units that stick together without being tethered by chemical bonding. It is the behavior of amphipathic molecules (part hydrophobic, part hydrophilic), which tend to self-assemble in aqueous solution. In short, many molecules spontaneously aggregate into regular structures. Phospholipids aggregate into planar bilayers called membranes. The spontaneity of the process has been exploited to reconstitute membranes in the laboratory in the presence of membrane proteins. Two techniques, still used today, have been developed in the 1960s and early 70s. Both make use of a setup made of two Teflon half-chambers separated by a very small hole. In the first case, the hole is covered by a lipid solution (in decane) and the membrane will spontaneously form while the organic solvent slowly dilutes into solution leaving behind a phospholipid layer which at its thinnest part will be a molecular bilayer. The formation can be monitored electronically via capacitance measurements. The second technique makes use of the apposition of two individual monolayers of phospholipids that always form at the water surface. When covering the hole from both sides by water covered with such a monolayer, the two monolayers will get in contact through the hydrophobic fatty acid parts to form a molecular bilayer with the same electronic properties as a bilayer formed by decane thinning. 

The following pictures show one of the earlier two chamber systems used by M. Montal at the University of California, San Diego.

The central partition can be lowered and risen relative to the water surface on each side. 

When moving the partition below the water surface, each side will be covered by a monolayer which will then interact to form a phospholipid bilayer. 

(Photos Lukas Buehler)

A refined version of the technique makes use of micrometer holes which are burned or cut into an extremely thin Teflon foil. H. Schindler developed the technique to use completely solvent free membranes to provide optimal recreation of a biological membrane. 

(Teflon septum (upper panel) with cut hole, diameter about 100micrometer (lower panel); H. Schindler; Photos Lukas Buehler)

These Teflon septum can be fixed between two small half-chambers and each side of the partition can be filled with water and carefully spread lipid monolayer on the surface. Using tubing connected to syringes can be used to rise the water levels on both sides.

The forming molecular bilayer across the aperture in the Teflon septum can be used to study ion channel activity, i.e., single channel recordings like the one shown above. 

Teflon half chamber with membrane in Faraday cage and inserted electrodes ready for current measurements.

H o m e
Copyright © 2000-2003 Lukas K. Buehler