Hrp linked anti rabbit 1gg

For the immunoblot analysis the same protein extraction was followed as for first dimensional gel electrophoresis (protocol given above). After electrophoresis, the gels were transferred to 0.45 µM nitrocellulose membrane (Sigma-Aldrich). After protein transfer, the blots were blocked with 5 % nonfat dry skimmed milk or bovine serum albumin (Sigma-Aldrich). After washing with TBS, the blots were incubated with monoclonal primary antibodies 1:1000 dilution of anti-SOD (Cell Signaling #2770, Danvers, MA, USA) for superoxide dismutase, and 1:1000 dilution of anti-APX/L (Cell signaling #AS08 368) for ascorbate peroxidase overnight at 4 °C. The blots were treated with 1:1000 dilution HRP-linked anti-rabbit 1gG (Cell Signaling #7074) for 1 h as a secondary antibody. Chemiluminescence reactions were performed with super signal west pico chemiluminescent substrates (Cell Signaling SignalFire^TM ECL Reagent #6883) and images were captured on ChemiDoc imaging system (BioRad, Hercules, CA, USA).

For western blotting, the above-mentioned protein extraction method was followed for first dimension gel electrophoresis (SDS-PAGE) for all the leaf samples. SDS-PAGE (1D) was performed, the protein gels were transferred to a PVDF membrane (Bio-Rad, Hercules, CA, USA) and western blot was performed according to Muneer et al. [39 (link)]. Once the protein was transferred to the membrane, the blocking solution that was prepared using 5% non-fat dry skimmed milk was used to block the blot immediately after the transfer was done, and side by side, another blot, which was Coomassie stained, was used as loading control. Incubation with polyclonal primary antibody of dilution factor 1:1000 of anti PsaA (Agrisera # AS06 172) for PsaA was conducted for 1:30 h. After primary antibody incubation, the bot was incubated for another one hour with a secondary antibody with a 1:1000 dilution factor identified as HRP-linked anti-rabbit 1gG (Cell Signaling #7074). Image analysis was performed using super signal west Pico chemiluminescent substrates (Cell Signaling Signal Fire ECL Reagent #6883) on a ChemiDoc imaging system (Bio-Rad, Hercules, CA, USA).