Horseradish peroxidase conjugated 6 his tag antibody

IPTG-induced cells (1 mL) were collected by centrifugation and then resuspended in 100 µL of ddH₂O. The whole-cell or purified samples were mixed with 1:1 (v/v) 1× Loading Buffer (100 mM Tris-HCl, pH 6.8, 3.2% (w/v) SDS, 0.04% (w/v) bromophenol blue, 16% (v/v) glycerol, and 40 mM DL-dithiothreitol). The samples were placed in a boiling water bath for 10 min and then cooled to room temperature. After centrifugation at 10,000 ×g for 10 min, 10 μL of each sample was subjected to SDS-PAGE and western blotting using the methods described in our previous work [38 (link)]. Horseradish peroxidase-conjugated 6×His-tag antibody (Abmart, Shanghai, China) was used to detect the His-tagged proteins. A B. abortus monoclonal antibody (Thermo Fisher Scientific, Waltham, MA, USA) was diluted 1:100 and used to detect polysaccharides of B. abortus in the glycoproteins. A Y. enterocolitica O:9 monoclonal antibody (Fitzgerald, Acton, MA, USA) was diluted 1:200 and used to detect Y. enterocolitica O:9 polysaccharide in the glycoproteins.

Western blot was performed as described previously [27 (link)]. Horseradish peroxidase–conjugated 6× His Tag antibody (Abmart Shanghai Co., Ltd., Shanghai, China) was used to detect proteins with a 6× His tag. If there were more nonspecific bands, the 6× His antibody was incubated with E. coli W3110 cell lysate for 1 h before detection. The antibodies against K. pneumoniae serotypes O1 and O2 were obtained by immunizing Japanese white rabbits with K. pneumoniae strain 355 and K. pneumoniae strain 041 whole bacteria, respectively, which were used to detect the glycan fraction of glycoproteins. Horseradish peroxidase-conjugated goat anti-rabbit IgG (Transgen Biotech, Inc., Beijing, China) was used as the secondary antibody.