Gold cantilever nsg01

A total of 3 μL of purified HEV-3 ORF2 110–610_6his recombinant protein was adsorbed onto glow discharged Carbon-Formvar-coated copper grids for 60 s. The grids were then washed by floating on water droplets, followed by staining with 2% (w/v) uranyl acetate (UA) for 20 s. Particles were imaged using an FEI Tecnai G2 20 (FEI, Hillsboro, OR, USA) electron microscope operating at 200 kV. An Integra Prima microscope and Nova SPM software (NT-MDT, Moscow, Russia) were used for atomic force microscopy. Scanning was performed in semi-contact mode, using gold cantilever NSG01 (NT-MDT). The protein sample was applied to a sapphire substrate coated with mica and dried at room temperature. PBS was used as a negative control. Cryo-EM grids were prepared as described by Byrne et al., 2019 [49 (link)].

The particles formed by the recombinant protein after refolding were examined using a transmission electron microscope and atomic force microscope. Electron microscopy was performed on a JEM 1400 instrument (JEOL, Tokyo, Japan). The purified proteins were placed on carbon-formvar-coated copper grids (TED PELLA, Redding, CA, USA) and stained with 1% (w/v) uranyl acetate in methanol. Particle sizes (n = 20) in digital photographs were determined using the ImageJ software [62 (link)].
Atomic force microscopy was performed using an Integra Prima microscope and Nova SPM software (NT-MDT, Moscow, Russia). The scanning was performed in the semi-contact mode using gold cantilever NSG01 (NT-MDT). Particle sizes (n = 20) were determined using the NT-MDT Nova v. 1.06.26 software supplied with the instrument.