| Popis: |
The nystatin-ergosterol (N/E) method is described and reviewed. Using this procedure, an experimenter can promote and detect fusion of vesicles with planar lipid bilayers. N/E fusion provides a straightforward mechanism to reconstitute any membrane protein into planar lipid bilayers. Once reconstituted, it is easy to determine the ion selectivity, transport rate, voltage dependence, and kinetics of any conductance caused by the membrane protein. Fusigenic N/E vesicles are made with a mixture of phospholipids, ergosterol, and nystatin. Vesicle size can be adjusted either with sonication or with polycarbonate filters. The best vesicles contain approximately 20 mol% ergosterol, are approximately 200 nm in diameter, and are in a solution containing approximately 50 micrograms/ml nystatin. Vesicle fusion requires an osmotic gradient and delivery of vesicles to the bilayer. Vesicle delivery is increased by (1) stirring of the chamber that contains vesicles, (2) larger bilayers, and (3) bilayers that are face-flush with the vesicle-containing solution. Because constant stirring is critical for delivery of vesicles to the bilayer, a system that allows simultaneous stirring and sensitive electrical measurements is desirable. The main strength of the bilayer technique has always been that the experimenter has control over the milieu of the membrane system. The N/E fusion technique adds to this strength by controlling fusion of vesicles to the bilayer, thus allowing the quantitative transfer of isolated proteins from vesicle to bilayer. The techniques and calculations necessary for successful quantitative reconstitution are given in detail. |
