Whole cell expressed in Sf9 cells, the soluble fraction obtained after lysis, and purified hEC-SODf and hEC-SODtr were mixed with 5× sample buffer and resolved by 12% SDS-PAGE at 120 V for 2 h. Western blot analysis was performed as described by Yun et al. (2005). Monoclonal anti-FLAG M2 alkaline phosphatase antibody (Sigma), and SIGMAFAST BCIP/NBT (Sigma) were used for visualization. A mouse monoclonal primary antibody was used at a dilution of 1:2000 to detect the FLAG-tagged recombinant hEC-SOD.
Anti-Superoxide Dismutase 3 antibody (abcam® - ab21974) was used to detect native form of hEC-SOD before and after DTT titration. A secondary anti-rabbit antibody was used at a dilution of 1:5000 to detect the recombinant hEC-SOD. The 12% Native PAGE was run at 90V for 3 h at 4°C to avoid denaturing the protein.
Anti-Superoxide Dismutase 3 antibody (abcam® - ab21974) was used to detect native form of hEC-SOD before and after DTT titration. A secondary anti-rabbit antibody was used at a dilution of 1:5000 to detect the recombinant hEC-SOD. The 12% Native PAGE was run at 90V for 3 h at 4°C to avoid denaturing the protein.