Nanoscope iiia microscope

Surface morphology was analysed by using Atomic Force Microscopy. Images were obtained with a Nanoscope IIIA microscope (Digital Instruments, Veeco Metrology Group, Chadds Ford, PA, USA) using the Tapping mode. Pore size distribution was analysed by the extended bubble point method, or Air Displacement Porometry (ADP), using a Coulter ^® Porometer-II manufactured by Coulter Electronics (Porometer, Aptco Invest, Dulles, VA, USA) [36 (link)]. Samples were first wetted with an electronic liquid FC-43 (Fluorinert^TM, 3 M, St. Paul, MN, USA) of low surface tension (γ 1.6 × 10⁻² N m⁻¹), low vapour pressure (192 Pa) and low reactivity, that can be assumed to fill all the pores given a zero contact-angle with the membrane material. The wetted sample was subjected to increasing pressure, applied by a compressed clean and dry air source. The pressure range for PTFE 0.22 membrane was 0.09–0.60 µm.

Atomic force microscopy (AFM) images (512 × 512 pixels with a scan size of 2 µm) were collected in air with a Nanoscope IIIA microscope (Digital Instruments, Santa Barbara, CA, USA) operating in tapping mode and equipped with the E scanner. Commercial silicon cantilevers (MikroMasch, Tallinn, Estonia) with a nominal tip radius of 5 nm were used. In the first set of experiments, 21 μM GgUox or DrUox pre-incubated with 0 or 300 μM βME was mixed with 2.1 mM urate in 100 mM potassium phosphate buffer, pH 7.6, and incubated at RT for 60 min. In a second set, 7 μM GgUox or DrUox pre-incubated with 0 or 100 μM βME was mixed with 100 μM H₂O₂ in 100 mM potassium phosphate buffer, pH 7.6, and incubated at RT for 60 min. All samples were diluted in deposition buffer (4 mM HEPES pH 7.4, 10 mM NaCl, 2 mM MgCl₂) to a final protein concentration of 100 nM, and 20 µl were deposited onto freshly cleaved mica that had been previously incubated with 50 µL MilliQ water for 5 min, and with 50 µl deposition buffer for 3 min. After 5 min incubation, the excess sample on the mica surface was removed by rinsing with 50 µl MilliQ water and drying under a nitrogen flux.

Purified virus was adsorbed onto the surface of freshly cleaved mica for 1 min, after which the solution was carefully removed with filter paper. This substrate was immediately placed on to a drop of double-distilled water (this procedure was repeated twice), and the surface was dried under air flow. This sample preparation method was used to eliminate any remaining salts and minimize any risk of aggregation during drying. AFM analysis was performed on these samples using a Nanoscope IIIA microscope (Digital Instruments, USA) operating in tapping mode with a typical scan rate of 1 Hz. The measurements were performed in air in tapping mode using sharp silicon cantilevers (NT-MDT, Russia) with a guaranteed tip radius of 10 nm.