Horseradish peroxidase hrp conjugated goat anti rabbit igg antibody

The protein concentrations were quantified using a BCA Protein Assay Kit (#CW0014S, CwBiotech, Taizhou, China) in line with the manufacturer’s instructions. After the addition of 6× SDS loading buffer, the protein samples were boiled for 10 min. Proteins were separated by 12.5% SDS-PAGE gels and transferred to PVDF membranes. Then, the blots were probed with anti-LsSP1 serum or anti-OsOryzain serum diluted at 1:5000, followed by additional incubation with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG antibody (1:10,000, #31460, ThermoFisher Scientific). Images were acquired by an AI 680 image analyzer (Amersham Pharmacia Biotech, Buckinghamshire, UK). The band intensities in immunoblot analyses were quantified using ImageJ software v1.53e (https://imagej.nih.gov/). To monitor the equal protein loading, samples were further stained with Coomassie brilliant blue (CBB). The full scan results of blots and gels were provided in Supplementary Fig. 23 and Source Data file.

The recombinant L. johnsonii-COE was cultivated statically in MRS medium containing 10 μg mL⁻¹ chloromycetin at 37 °C. The bacteria were harvested by centrifugation at 12,000× g for 1 min, and washed with sterile phosphate-buffered saline (PBS) twice. The bacteria were lysed by ultrasound treatment, pelleted by centrifugation, and the sediment was analyzed by western blotting as previously described [15 (link)], using the rabbit anti-COE monoclonal antibody (prepared in our laboratory) and horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG antibody (Thermo Fisher, San Jose, CA, USA) as primary and secondary antibodies, respectively.