MERS-CoV Protein Detection by Western Blot

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).