Hrp conjugated goat anti human igg h l

Five μl of each LPS was separated by electrophoresis as described above. LPS was transferred to 0.2 μm polyvinylidene difluoride (PVDF) membranes using the Trans-Blot Turbo blotting system (Bio-Rad) with a high-molecular-weight (MW) program (1.3A up to 25 V for 10 min). The membranes were blocked overnight in 5% BSA in PBS plus 0.1% Tween 20 (PBST), then incubated with 1 μg/ml KM467 or polyclonal anti-O25 typing sera (Abcam, diluted 1:10) in 0.5% BSA in PBST for 2 h at RT. The membranes were washed 3 × 10 min in PBST, then incubated with 0.1 μg/ml HRP-conjugated Goat Anti-Human IgG H&L or HRP-conjugated Goat Anti-Rabbit IgG H&L (Abcam) in 0.5% BSA in PBST for 1 h at RT. The membranes were washed 6 × 10 min in PBST, then developed with SuperSignal West Pico PLUS (Thermo Scientific).

12% SDS‐PAGE was used for electrophoreses and Western Blot. The PBS buffer and irrelevant protein (cellular protein) were used as controls. The serum at 1:100 dilution from COVID‐19 patients used as primary antibody was incubated at 37℃ for 2 h, then washed with TBST 3 times and 5 min each time. HRP‐conjugated Goat anti‐human IgG(H + L) (Abcam) at 1:50,000 dilution was used as the secondary antibody that was incubated at 37℃ for 2 h, then the TBST washed 5 times and 5 min each time. Finally, Clarity western ECL substrate (Bio‐Rad) was used to visualize the band.