Fresh DOPC:DOPS (1:1) SUVs were unrolled on freshly cleaved mica (∅ 1.2 cm) in HBS-Ca (10 mM HEPES, 150 mM NaCl, 2 mM CaCl2, pH 7.5), by covering the mica entirely with 0.2 mg ml−1 SUVs, followed by a 20-min incubation at 30 °C, and washing twice with HBS-Ca.
The samples were imaged immediately in HBS at ambient temperature using an Asylum Research MFP-3D Bio instrument. TR800PSA cantilevers (Olympus; spring constant (k) range 0.59–0.68 N m−1, resonance frequency 77 kHz) were used in alternative current (AC) mode. Square 256 × 256 pixel scans were acquired from areas between 5–20 µm, using a 90° scanning angle and a 0.6–0.8 Hz scan speed. The resulting scan images were processed in Igor Pro 6.37.
After confirming the presence of lipid bilayers, 1.8–10 µM P0ct was added onto the bilayer samples in HBS. After a 15-min incubation period at ambient temperature, the bilayers were washed twice with HBS, and imaged as above. For each protein concentration, 2 samples were prepared and scanned with identical results. At least 3 different parts per sample were scanned to gain an insight into any sample heterogeneity.
The samples were imaged immediately in HBS at ambient temperature using an Asylum Research MFP-3D Bio instrument. TR800PSA cantilevers (Olympus; spring constant (k) range 0.59–0.68 N m−1, resonance frequency 77 kHz) were used in alternative current (AC) mode. Square 256 × 256 pixel scans were acquired from areas between 5–20 µm, using a 90° scanning angle and a 0.6–0.8 Hz scan speed. The resulting scan images were processed in Igor Pro 6.37.
After confirming the presence of lipid bilayers, 1.8–10 µM P0ct was added onto the bilayer samples in HBS. After a 15-min incubation period at ambient temperature, the bilayers were washed twice with HBS, and imaged as above. For each protein concentration, 2 samples were prepared and scanned with identical results. At least 3 different parts per sample were scanned to gain an insight into any sample heterogeneity.