Anti mouse igg conjugated with horseradish peroxidase

For Western blot experiments, R. sphaeroides cultures were grown under semi-aerobic and ¹O₂ stress conditions as described above. Samples were taken at the indicated time points. Cells were broken by sonication and cell debris removed by centrifugation. Total protein extracts were separated on 10% PAA-SDS gels and transferred to nitrocellulose membranes (Whatman). Proteins were stained and fixed with Ponceau S (Sigma-Aldrich) and de-stained with sodium hydroxide. Membranes were blocked for 1 hour at room temperature with blocking buffer (1x TBS) containing 5% (w/v) milk powder (Roth). After blocking, the purified primary antibody α-GFP, diluted 1:4000 in blocking buffer, was added to the membrane and incubated for 1 hour. After washing the membrane 3 times for 5 min in 1x TBS buffer, the secondary antibody (anti-mouse IgG conjugated with horseradish peroxidase, produced in goat, Sigma-Aldrich) was added (diluted 1:5000 in blocking buffer) and the membrane further incubated for 1 hour at room temperature. The membrane was washed 3 times with 1x TBS for 5 minutes. Western blots were developed using the Lumi-Light Western Blotting Substrate 1 and 2 (Roche) and analyzed on a Chemiluminescence-Imaging System (Fusion-SL4; Peqlab).

Levels of eNOS, nNOSα monomer and dimer were quantified by western blotting via low temperature (LT)-PAGE using vascular, stomach and colon specimens from control and infected mice as described previously [25 (link), 26 ]. Briefly, LT-SDS-PAGE was performed on ice; 30 μg of protein in standard Laemmli buffer was incubated at 0°C for 30 min and then separated using a 6% separating gel. All gels and buffers were pre-equilibrated to 4°C prior to electrophoresis; the buffer tank was also placed in an ice-bath during electrophoresis to maintain the gel temperature below 15°C. A polyclonal antibody specific to nNOSα (1:1,000 dilution) (Zymed Laboratories, Grand Island, NY, USA), a monoclonal antibody specific to eNOS (1:500) (BD Biosciences, San Jose, CA, USA), anti-rabbit IgG and anti-mouse IgG conjugated with horseradish peroxidase (1:5,000) (Sigma Chemical, St. Louis, MO, USA) were used as the primary and secondary antibodies, respectively.