Single channel recording of OmpG was similar to the previous study.50 (link) Briefly, experiments were performed in an apparatus containing two chambers separated by a 25 μm thick Teflon film. An aperture of approximately 100 μm diameter had been made near the center of the film with an electric spark. The aperture was pretreated with a hexadecane/pentane (10% v/v) solution before each chamber was filled with buffers as indicated specifically. An Ag/AgCl electrode was immersed in each chamber with the cis chamber grounded. 1,2-Diphytanoyl-sn-glycerol-3-phosphocholine (Avanti Polar Lipids, USA) dissolved in pentane (10mg/ml) was deposited on the surface of the buffer in both chambers and monolayers formed after the pentane evaporated. The lipid bilayer was formed by raising the liquid level up and down across the aperture. OmpG proteins (~1 nM, final concentration) were added to the cis chamber and +200mV was applied to facilitate OmpG insertion. After a single OmpG pore inserted, the applied voltage was lowered to 50 mV for recording. OmpG proteins inserted in the planar lipid bilayer bi-directionally with its extracellular loops located at either cis or trans side. After 10 min recording, the orientation of the OmpG pore in the lipid bilayer was determined by analyzing the asymmetrical gating pattern at positive and negative potentials.61 (link) Streptavidin or antibodies were added to the cis or trans chamber depending on the pore orientation and the solution was stirred for 10 s. We define a positive potential as the potential of the chamber where the extracellular loops were exposed to is positive. Current was amplified with an Axopatch 200B integrating patch clamp amplifier (Axon Instruments, Foster City, CA). Signals were filtered with a Bessel filter at 2 kHz (unless otherwise stated) and then acquired by a computer (sampling at 50 μs) after digitization with a Digidata 1320A/D board (Axon Instruments).