Peroxidase conjugated secondary antibody goat anti rabbit igg

We detected the protein levels of the CON group, DSS group, and H-BBr group by using Western blot. Extracted tissue protein on ice, and the protein concentration was determined with a BCA protein determination kit and diluted to an equal concentration (2–4 μg). Protein was separated by 6, 8, 10, and 12% SDS-polyacrylamide gel electrophoresis. The membrane transfer time is proportional to the protein size. After sealing with 5% non-fat milk for 2 h at room temperature, the membrane was incubated overnight with cat primary antibody (Supplementary Table 3) at 4°C and acquired with peroxidase-conjugated secondary antibody goat anti-rabbit IgG (1:2,000, Cell Signaling Technology) for 2 h at room temperature. Finally, it detected the signal with X-ray films (TransGen Biotech Co., Beijing, China).

Protein content of samples were estimated by BCA reagent. Protein concentration in each sample was diluted to 20 μg/μL. Before the experiment, nitrocellulose membrane was activated by TBST (50 mM Tris, 0.5 M NaCl, 0.05% Tween 20, pH 7.4) for 20 minute at room temperature. Samples (1 μL) were spotted onto the membrane in 3 different dilutions (100x, 500x, and 1000x). The membrane was blocked in 5% nonfat dry milk in TBST for 1 hour at room temperature. The membrane was then incubated with an anti-nitrotyrosine primary antibody (Merck Millipore), which was diluted in 1% milk in TBST overnight at 4°C. After washing three times with TBST, the membrane was incubated with peroxidase-conjugated secondary antibody (goat anti-rabbit IgG, Cell Signaling Technology) diluted in 1% milk in TBST for 1 hour at room temperature. To visualize the antibody reaction, chemiluminescent substrates were added, and chemiluminescent signals were detected using a FluorChem FC2 Imager. Densitometric analysis was carried out using ImageJ software. Nitrotyrosine values were expressed in mol/μg protein calculated from the values of peroxynitrite-treated BSA, which were used as a positive control.