Horseradish peroxidase hrp conjugated secondary antibody goat anti human igg

To validate the antigenicity and protein purity of plant- and E. coli-based rNPs, we tested a serum sample positive for SARS-CoV-2 infection obtained from a patient in the convalescent phase. For a negative control, pooled negative sera were obtained from the healthy individuals prior to the COVID-19 pandemic. The proteins were separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by electroblotting of protein bands onto polyvinylidene difluoride (PVDF) membranes (Thermo Fisher Scientific, Germany). The blot was cut into strips and blocked with blocking buffer (phosphate-buffered saline with Tween 20 [PBS-T] containing 5% skimmed milk) for 1 h at room temperature followed by incubation with serum samples as primary antibodies (1:1,000 diluted in blocking buffer) at 4°C overnight. After a wash step in PBS-T for 10 min, the bound antibodies were detected using horseradish peroxidase (HRP)-conjugated secondary antibody goat anti-human IgG (Invitrogen, USA) at a dilution of 1:10,000 in PBS-T for 1 h at room temperature. The immunoprecipitated bands were developed using enhanced chemiluminescence reagents, and the membranes were scanned with an infrared imaging system. The expression and purity of both proteins were identified by SDS-PAGE gel stained with InstantBlue Coomassie protein stain (ab119211).

To validate the antigenicity and protein purity of plant- and E. coli-based rNPs, we tested a serum sample positive for SARS-CoV-2 infection obtained from a patient in the convalescent phase. For a negative control, pooled negative sera were obtained from the healthy individuals prior to the COVID-19 pandemic. The proteins were separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by electroblotting of protein bands onto polyvinylidene difluoride (PVDF) membranes (Thermo Fisher Scientific, Germany). The blot was cut into strips and blocked with blocking buffer (phosphate-buffered saline with Tween 20 [PBS-T] containing 5% skimmed milk) for 1 h at room temperature followed by incubation with serum samples as primary antibodies (1:1,000 diluted in blocking buffer) at 4°C overnight. After a wash step in PBS-T for 10 min, the bound antibodies were detected using horseradish peroxidase (HRP)-conjugated secondary antibody goat anti-human IgG (Invitrogen, USA) at a dilution of 1:10,000 in PBS-T for 1 h at room temperature. The immunoprecipitated bands were developed using enhanced chemiluminescence reagents, and the membranes were scanned with an infrared imaging system. The expression and purity of both proteins were identified by SDS-PAGE gel stained with InstantBlue Coomassie protein stain (ab119211).