Nbt bcip detection kit

Bacterial cell extracts were lysed in Lämmli buffer and separated by SDS-PAGE. For protein analysis, NuPAGE™ Novex™ 3–8% Tris-acetate protein gels (Thermo Fisher Scientific, Waltham, MA, USA) were used and either stained with Colloidal Blue (Thermo Fisher Scientific) or subjected to western blotting. For the analysis of glycolipids (LPS or LLO), NuPAGE™ Novex™ 12% Tris-acetate gels (Thermo Fisher Scientific) were used and extracts were additionally treated with Proteinase K (1 g/L) for 1 h at 37 °C after lysis in Lämmli buffer to suppress protein signals. For immunoblotting, samples were transferred using an iBlot® 2 Dry Blotting System (Thermo Fisher Scientific) followed by immunostaining. Polyclonal anti-Pseudomonas Exotoxin A antibody produced in rabbit (Sigma-Aldrich, Switzerland), a polyclonal rabbit anti-E.coli O25 antiserum (Denka Seiken, Tokyo, Japan), or custom made antisera were used for immunoblotting as described [17 (link)]. For signal visualization, secondary goat anti-rabbit IgG-horse radish peroxidase conjugate (Sigma-Aldrich) in combination with NBT/BCIP detection kit (Roche, Switzerland). Agglutination assays were performed as described previously (DebRoy et al. Dec).

The WT and c-di-GMP mutant strains were grown overnight in SCFM. To ensure equal amounts of proteins were loaded, cell cultures were adjusted to an OD₆₀₀ of 1.0 before taking equal volumes. To prepare the whole cell lysates, the cells were thawed, pelleted and resuspended in 2X SDS loading dye. After boiling, the whole cell lysates were separated by 12% SDS-PAGE to separate protein and transferred onto PVDF membrane. Then, the FliC protein production level was detected with the FliC specific primary polyclonal anti-flagellin antibody by incubating the membrane for 45 min at 4°C on the shaker. The polyclonal anti-flagellin antibody was raised against B. pseudomallei in rabbit (kindly gifted by Dr. David Speert). The membrane was then incubated with the secondary specific rabbit antibody tagged with alkaline phosphatase (Sigma-Aldrich, USA) for 45 min on a shaker. The ratio of primary and secondary antibodies dissolved in blocking buffer were 1:20,000 and 1:30,000, respectively. Finally, the FliC-primary antibody complex and secondary antibody interaction were detected with an NBT/BCIP detection kit (Roche, USA).

The WT and mutant strains were grown overnight in SCFM, MOPS-glucose 20 mM and MOPS-glucose 5 mM with or without amino acids. To ensure equal amounts of protein were loaded, whole cell lysates were prepared from same volumes of cultures adjusted to an OD₆₀₀ of 0.5. To prepare the whole cell lysates, the cells were thawed, pelleted and resuspended in 2X SDS loading dye. After boiling, the whole cell lysates were run in a 12% SDS-PAGE to separate protein and transferred onto PVDF membrane. Then, the FliC protein expression level was detected with the FliC specific primary anti-flagellin antibody raised in rabbit (kindly gifted by Dr. David Speert) and the secondary specific rabbit antibody tagged with alkaline phosphatase (Sigma-Aldrich, USA). Finally, the FliC-primary antibody complex and secondary antibody interaction was detected with a NBT/BCIP detection kit (Roche, USA). The ImageJ software was used to analyse the relative band intensity of the Western blot bands and the fold change was calculated in reference to MOPS-glucose.