Sds page loading buffer 5

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).