Gegegnome xrq enhanced chemiluminescence ecl

SDS-PAGE loading buffer (non-reducing, 5×) (Cowin Bio., Guangzhou, China) was added to the RBD-6HB protein sample at a ratio of 1:4 and centrifuged at 3000 rpm for 30 s to prepare a non-reduced RBD-6HB protein sample. Meanwhile, SDS-PAGE loading buffer (5×) (Beyotime, China) was added to the protein sample at a ratio of 1:4 after 10 min of boiling water, then centrifuged at 3000 rpm for 30 s to prepare a reduced RBD-6HB protein sample. Finally, we obtained a non-reduced RBD-6HB protein sample and a reduced RBD-6HB protein sample. After centrifugation, an appropriate amount of supernatant was taken with a micropipette and directly added to the 10% SDS-PAGE gel sample wells. After a transfer by semi-dry transfer method, block with 5% skim milk at room temperature for 2 h, incubate with anti-RBD mouse polyclonal antibody, and wash with TBST. After that, the membrane was incubated with HRP-labeled Anti-Rabbit IgG (H + L) (1:5000, Beyotime, China) for 1 h at room temperature, washed with TBST 3 times, and finally, the band was developed using the GEGEGNOME XRQ enhanced chemiluminescence (ECL) (Thermo Fisher Scientific, Waltham, MA, USA).

Western blot was performed according to the protocol described by Xu et al. (Xu et al., 2021 (link)). Briefly, cell lysate or protein solution was loaded (1:5) and separated via 10% SDS-PAGE. Then, the protein was transferred onto the PVDF membrane, blocked with 5% skim milk for 1 h at room temperature, followed by incubating with the convalescent serum of the COVID-19 patient (1:1000) or Influenza A M1 (1:1000, GeneTex, USA) for 2 h at room temperature. After that, the membrane was incubated with HRP-labeled Goat Anti-Rabbit IgG (H+L) or HRP-labeled Goat Anti-Mouse IgG (H+L) (1:3000, Beyotime, China) for 1 h at room temperature. Subsequently, the band was developed using the GEGEGNOME XRQ enhanced chemiluminescence (ECL) (Thermo Fisher SCIENTIFIC, USA).

rBV-SARS-CoV2-RBD-6HB was inoculated into sf9 cells, and the cells were collected after 72 h of culture. After adding IP cell lysate (Beyotime, Shanghai, China), the cells were sonicated and centrifuged at 12,000 rpm for 3 min at 4 °C to collect the supernatant. After adding a certain volume of 5 × SDS to the supernatant, SDS-PAGE electrophoresis was performed after 10 min of boiling water bath. After a transfer by semi-dry transfer method, block with 5% skim milk at room temperature for 2 h, RBD and RBD-6HB incubate with anti-RBD mouse polyclonal antibody;6HB incubate with Rb pAb to Strep-tag (1:1000, Abcam, Cambridge, UK, ab76949), then wash with TBST. After that, the membrane was incubated with HRP-labeled Anti-Rabbit IgG (H+L) (1:5000, Shanghai, Beyotime, China) or HRP-labeled Anti-Mouse IgG (H + L) (1:5000, Beyotime, Shanghai, China) for 1 h at room temperature, wash with TBST 3 times, and finally, the band was developed using the GEGEGNOME XRQ enhanced chemiluminescence (ECL) (Thermo Fisher Scientific, Shanghai, China).