PLY mutant pores were compared to wildtype protein formed on planar lipid layers or cholesterol-containing liposomes prepared as above. The liposome mixture was diluted to a final concentration of 0.5 mg · ml−1 and 25 µg · ml−1 PLY was added. Planar lipid bilayers and PLY were incubated in Teflon well plates. 25 µg · ml−1 PLY in reaction buffer (150 mM NaCl, 50 mM Tris-HCl pH 7.0, 5 mM β-mercaptoethanol) was transferred into a well and overlaid with a droplet of a 0.5 mg · ml−1 DOPC/cholesterol solution (1:1 molar ratio) in chloroform. Planar lipid layers formed upon chloroform evaporation. Wells were incubated for 30 min at 37°C, after which PLY-containing membranes were transferred to EM grids and stained with 1% (wt/vol) uranyl acetate. Negatively stained specimens were examined in an FEI Tecnai Spirit electron microscope at an acceleration voltage of 120 kV. Images were recorded on a 2 K Gatan CCD camera at a magnification of 25,000x–45,000x and ∼1.0–1.5 µm underfocus.