Ethylene diamine tetraacetic acid (edta)

For immunoprecipitation pre-treatment using the sample with protein quantification, homogenization was performed after adding 0.5 M EDTA (Duksan, Korea), lysis buffer (Gendepot, R4200-100, USA), and phosphatase inhibitor 100 × (Gendepot, P3200-001, USA) to the sample. After adding 200 μg/ml each of Cu-Zn-SOD and GPx, which are the primary antibodies, into the homogenized sample of 1ml, the mixture was left over-night (12 hours) at 4℃. After inserting 30 μl of Beads (Dynabeads, Protein A) and 1 × PBS of 500 μl in the sample left over-night and incubating at 4℃ for 1 hour, the incubated sample was washed 3 times for 5 minutes with 1 × PBS using a magnetic particle concentrator. 1 × PBS of 50 μl was added to the washed sample, and the samples were quantified by mixing with Laemmli sample buffer. Western blotting for the Cu,Zn-SOD (Santa cruz, sc-11407, USA) and GPx (Santa cruz, sc-22145, USA), which are primary antibodies, and their secondary antibodies were performed according to the manufacturers’ instructions. Identification of the band was developed on X-ray film after illuminating a membrane using an enhanced chemiluminescene (ECL) kit. The density of the developed band was calculated by band/β-actin using image j software.