Amicon ultra 0.5 ml centrifugal filters ultracel 3k

Freshly purified NP was dialyzed against TBS overnight using an EasySep dialysis membrane MD-014–50 (Tomy Seiko) according to the manufacturer’s instructions. The protein was then concentrated using Amicon Ultra-0.5 mL Centrifugal Filters, Ultracel-3 K (Merck Millipore). Concentrated CCHFV NP samples (5 µl) were applied on a collodion-coated copper grid (Nisshin EM) for 5 min then the excess sample was absorbed using filter paper. A 20 µl drop of 2% uranyl acetate (UA) solution was applied for 10 min. The grid was then treated with new drops of UA solution 2 times for each 1 min. EM images were observed using a Hitachi H7650 transmission electron microscopy (TEM) system (Hitachi High Technology Corporation). For immunogold staining, the rabbit antiserum to CCHFV NP and 5 nm gold-labeled goat anti-rabbit IgG (Biorbyt LLC) were used, followed by 2% UA staining.

The indicated cells were transfected with the indicated plasmids and cultured for 2 days in 0.1% FBS-containing medium under normoxic conditions. Both cell lysates harvested with CelLytic M (MilliporeSigma) and culture medium were subjected to Western blotting using anti-myc epitope tag mouse monoclonal antibody (1000-fold dilution; Cell Signaling Technology, clone 9B11, catalog 2276) for the detection of exogenously expressed SPINK1 and its derivatives and anti-human β-actin mouse monoclonal antibody (200-fold dilution; Santa Cruz, clone AC-15, catalog Sc-69879) as primary antibodies, anti-mouse IgG HRP-linked whole Ab (5000-fold dilution; GE Healthcare Bioscience) as secondary antibody, and ECL Prime Western Blotting Detection Reagents (GE Healthcare Bioscience) for detection. The culture media were 17 times concentrated using Amicon Ultra-0.5 mL Centrifugal Filters, Ultracel-3K (Merck Millipore) before Western blotting according to the manufacturer’s instructions.