Horseradish peroxidase hrp conjugated goat anti rabbit or mouse igg

Bacterial cells were lysed using 1× loading buffer and then heated at 95°C for 10 min before SDS-PAGE. Total proteins were examined by 12% SDS-PAGE and then electrotransferred onto a polyvinylidene fluoride (PVDF) membrane (Millipore). The membranes were blocked with 5% milk in phosphate-buffered saline plus Tween (PBST) at 25°C for 1 h, followed by incubation overnight at 4°C with polyclonal antibody to YdiU (1:2,000 dilution) or GapA (1:10,000 dilution) in PBST. After three washes with PBST, the membranes were then incubated at 25°C for 1 h with horseradish peroxidase (HRP)-conjugated goat anti-rabbit or mouse IgG (Abcam) diluted 1:10,000 in PBST. Finally, the membranes were incubated with a chemiluminescent substrate (Immobilon Western HRP substrate; Millipore) and detected using a FluorChem imager (Uvitec).

Bacterial total proteins were treated at 95°C for 10 min and were separated using 12% SDS-PAGE. The proteins were then electrotransferred onto a polyvinylidene fluoride (PVDF) membrane (Millipore, Bedford, MA). The membrane was blotted with polyclonal antibodies against YdiU, FlhDC, FliC, or GapA (internal) after blocking with 5% milk in phosphate-buffered saline plus Tween (PBST) and then incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit or -mouse IgG (Abcam) diluted in PBST. Finally, the signals were detected with chemiluminescent substrate (Immobilon Western HRP substrate; Millipore) using a FluorChem imager (UVITEC). The antibody against FliC used in this study is a polyclonal antibody purchased from Abcam (ab93713). The antibodies against YdiU, FlhDC, and GapA were prepared by Dia-An Biotech, Inc. (Wuhan, China), as described before (30 (link), 35 (link)). For Fig. 2, the FliC levels relative to that of GapA were quantified in grayscale in three independent experiments. The gray values were gathered using ImageJ.