Hi qras gel n

Cell-free synthesized proteins or cell culture supernatants containing inactivated MERS-CoV were mixed with an equal volume of 2X SDS sample buffer [125 mM Tris-HCl (pH 6.8), 4% SDS, 20% glycerol, 10% 2-mercaptoethanol and 0.01% bromophenol blue] and heated at 100°C for 5 min. After separation by 12.5% or 15% SDS-PAGE using Hi-QRAS Gel N (Kanto Chemical, Tokyo, Japan), the proteins were electrotransferred onto an Immobilon-P PVDF Transfer Membrane (Millipore, Bedford, MA, USA) as described previously (Nishi et al., 2014 (link)). The membrane was blocked in Tris-buffered saline (TBS) containing 2% (w/v) skim milk for 30 min, and then incubated for 1 h with anti-MERS-NP mAbs or anti-His polyclonal antibody (GTX115045; GeneTex, Irvine, CA, USA) in TBS containing 0.1% (v/v) Tween 20 (TBS-T; 1:1000 dilution) and 0.4% (w/v) skim milk. After three washes with TBS-T, the membrane was incubated for 60 min in PBS containing HRP-conjugated goat-anti mouse or rabbit IgG antibody (1:10000 dilution; GE Healthcare). After an additional three washes in TBS-T, proteins were detected with SuperSignal West Dura Extended Duration Substrate (Thermo Fisher Scientific, Rockford, IL, USA) or Immobilon (Millipore, Bedford, MA, USA) on a Lumi-Imager F1 (Roche Diagnostics, Basel, Switzerland).

Bovine Hb was purified from fresh bovine blood purchased from Tokyo Shibaura Zouki Co. Ltd. Purification procedures of native CSA from canine plasma are presented in Supplementary Materials. All other reagents were purchased from commercial sources as special grades and were used without further purification. Deionized water (18.2 MΩ·cm) was prepared using water purification systems (Elix UV and Milli Q Reference; Millipore Corp.). SDS-PAGE and Native-PAGE were performed using an electrophoresis power supply (EPS 301; GE Healthcare UK Ltd.) and 5–20% polyacrylamide precast gradient gel (Hi-QRAS gel N; Kanto Chemical Co. Inc.). Isoelectric focusing (IEF) was conducted using an electrophoresis power supply (EPS 601; GE Healthcare UK Ltd.) and IEF protein gels (Novex pH 3–10; Thermo Fisher Scientific Inc.). The UV-visible absorption spectra were recorded using a UV-visible spectrophotometer (8543; Agilent Technologies Inc.) connected with a temperature control unit (89090A; Agilent Technologies Inc.). Circulation dichroism (CD) spectra were recorded using a spectrophotometer (J-820; Jasco Corp.). Mass spectra were obtained using a MALDI–TOF mass spectrometer (autoflex equipped with a pulsed N₂ laser (337 nm); Bruker Daltonics K.K.). As the matrix, 0.1% sinapic acid, 0.05% trifluoroacetic acid, and 50% acetonitrile solution were used.

Cell lysates samples in SDS sample buffer were loaded onto 10%–20% SDS-PAGE using Hi-QRAS Gel N (Kanto Chemical, Tokyo, Japan), the proteins were electrotransferred onto an Immobilon-P PVDF Transfer Membrane (Millipore, Bedford, MA, USA). Immunoblotting using anti-SARS-CoV-2 NP mAbs, anti-His monoclonal antibody, or anti-DDDDK (FLAG) tag antibody (MBL, Aichi, Japan) was performed as previously described²¹ (link). For immunostaining analysis, cells were fixed with 4% paraformaldehyde and were permeabilized with PBS 0.5% Triton X-100 in PBS. After blocking with PBS containing 3% BSA, cells were stained with hybridoma culture supernatants (1:20 dilution) and Alexa Fluor568-conjugated secondary antibodies (Thermo Fisher Scientific). Cells were mounted using VECTASHIELD mounting medium with DAPI (Vector laboratories, CA, USA). For immunohistochemical analysis, formalin-fixed and paraffin-embedded tissue sections from the autopsy lung were de-paraffinized and rehydrated. After blocking with 5% goat serum, the sections were incubated with developed mAb #98 (1:20 dilution). After washing, the sections were incubated with the linker and then the polymer peroxidase-conjugated anti-mouse IgG solution (EnVision™ FLEX+; DAKO, Ely, UK), and then visualized with 3,3-diaminobenzidine (DAKO) and counterstained with hematoxylin.