Ecl prime immunoblotting detection kit

Salmonella TH12415 cells harboring an appropriate plasmid were grown exponentially in 30 ml L-broth containing 100 μg ml⁻¹ ampicillin at 30 °C with shaking. The cells were harvested, resuspended in 3 ml PBS, and sonicated. After the cell debris was removed by low-speed centrifugation, the cell lysates were ultracentrifuged (100,000 × g, 60 min, 4 °C). After carefully removing the soluble fractions, membranes were resuspended in 300 μl of sodium dodecyl sulfate (SDS) loading buffer [62.5 mM Tris-HCl, pH 6.8, 2% (w/v) SDS, 10% (w/v) glycerol, 0.001% (w/v) bromophenol blue] containing 1 μl of 2-mercaptoethanol and heated at 95 °C for 3 min. Proteins were separated by SDS–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes (Cytiva) using a transblotting apparatus (Hoefer). Then, immunoblotting with polyclonal anti-FliF or anti-FliN antibody was carried out. The primary antibody was diluted 10,000-fold and the secondary antibody was diluted 5000-fold using TBS-T buffer (20 mM Tris-HCl, pH 7.5, 500 mM NaCl, 0.1%(v/v) Tween-20). An ECL prime immunoblotting detection kit (GE Healthcare) was used to detect target bands. Chemiluminescence signals were detected by a Luminoimage analyzer, LAS-3000 (GE Healthcare). Three independent experiments were performed.