2.6. Western blotting
To analyze reactivity of peptide-adsorbed sera with SARS-CoV-2 RBD and
NP proteins, 2 µg of each protein in native or 1% 2-ME reduced form
were subjected to10%. Polyacrylamide gel in SDS sample buffer. Proteins
were separated by electrophoresis at 100 V for 1h, then transferred to a
0.45 μm hydrophilic polyvinylidene fluoride (PVDF) membrane at 110 V for
1.5 hours. Successful transfer and equal protein loading were confirmed
by Ponceau S (Sigma Aldrich) (0.1% Ponceau S in 5% acetic acid) and
Coomassie Brilliant Blue (Sigma Aldrich) staining (0.1% Brilliant blue
in 50% methanol, 10% acetic acid, 40% dH2O) on PVDF membrane and
polyacrylamide gel, respectively. Membranes were blocked in 5% skimmed
milk in PBS overnight at 4°C, after de-staining with H2O. Subsequently,
the membranes were incubated with serum samples diluted in blocking
solution for 45 minutes at room temperature (RT). After washing for 5
times with PBST, membranes were incubated with secondary HRP-conjugated
mouse monoclonal anti-human IgG at RT for 45 minutes, followed by
washing 5 times for 5 minutes. Finally, positive signals were detected
by chemiluminescence ECL Prime (GE Healthcare, Sweden) solution.