*2.6. Western Blotting and Transmission Electron Microscopy (TEM) Analysis of VLPs*

Purified SUDV VLPs were processed and examined by Western blotting and transmission electron microscopy. For Western blotting, aliquots containing 10 μg of total protein were diluted with reducing buffer and denatured by heating at 95 ◦C for 10 min. Proteins were separated in 12% acrylamide gels, before they were transferred onto polyvinylidene fluoride (PVDF) membranes (Merck Millipore, Darmstadt, Germany) under denaturing conditions. For protein detection, two polyclonal antisera were used: mouse anti-SUDV GP polyclonal antisera and mouse anti-SUDV VP40 polyclonal antisera were mixed at a dilution of 1:1500 as a primary antibody for blotting; a goat anti-mouse IgG HRP-conjugated antibody (Bioss antibodies, Beijing, China) was used at a dilution of 1:5000 as a secondary antibody.

Negative staining transmission electron microscopy (TEM) was used to analyze the shape and size of purified SUDV VLPs. In short, 30 μL of sucrose gradient-purified SUDV VLPs were fixed for 15 min on carbon-coated formvar grids, grids were washed with 30 μL PBS and then treated with 1% phosphotungstate acid for 5 min. Grids were left to air dry and observed by using a HITACHI H-7650 transmission electron microscope.
