Production of a polyclonal antibody against Arabidopsis thaliana ascorbate peroxidase 2

Abstract

Ascorbate peroxidase (APX) catalyzes the reduction of H2O2 to water by utilizing ascorbate as the electron donor. Among the cytosolic APX, the Ascorbate peroxidase 2 (APX2) plays an essential role in response to environmental stress in Arabidopsis thaliana. However, the study to explore molecular regulation of APX2 is hampered due to the lack of reliable molecular assay tools. Here, we produced an APX2 polyclonal antibody using its recombinant protein as an antigen. The full-length APX2 cDNA was isolated from a heat-treated Arabidopsis. The cDNA was cloned into the pET28a expression vector and then expressed in Escherichia coli BL21. The Escherichia coli effectively expresses the APX2 protein with the addition of 0.5 mM IPTG at 30°C for four hours. The APX2 protein was purified from soluble fraction using an affinity Ni-NTA resin and then released from the complex resin using thrombin protease. The recombinant APX2 protein was successfully purified with a molecular size of approximately 28 KDa and then injected into rabbits to raise polyclonal antibodies. Western blot analysis showed that the antiserum specifically recognizes endogenous APX2 (28 KDa) from Arabidopsis with a high titer ratio. The produced antibodies could be used for molecular assay to explore the molecular regulation of the APX2.