Ecl plus detection reagent

The OxyBlot protein oxidation detection kit (Millipore-Sigma) was used to measure the level of protein oxidation. The DNP reaction mixture was prepared by adding 15 μg intestinal protein sample (7 μL), 3 μL of 15% SDS, and 10 μL of DNP solution. The mixture was kept at room temperature for 15 min, followed by addition of 7.5 μL of neutralization buffer. Samples were then separated on a 10% SDS-PAGE gel, transferred to nitrocellulose (Bio-Rad) and blocked with 5% non-fat milk for 1 h. After washing with 1X PBS, the membrane was incubated with primary antibody (1:150) overnight at 4°C and then secondary antibody (1:300) for 1h at room temperature. Membranes were incubated with ECL plus detection reagent (Bio-Rad) and scanned using a chemiluminescent scanner (Bio-Rad). Band densities in a given lane were analyzed using ImageJ and summated. Afterward, the membranes were incubated with 15% hydrogen peroxide for 30 min at room temperature and treated with anti-actin antibody. OxyBlot values were then normalized to actin for each sample.

The level of protein oxidation was measured with the OxyBlot protein oxidation detection kit (Millipore-Sigma, Burlington, MA, USA). Worms were collected from each condition, washed three times with S-basal and concentrated to 10 μl lysis buffer and frozen to -80 °C for storage. Worms were homogenized with a TissueLyser II (Qiagen, Germantown, MD, USA). The DNP reaction mixture was made by adding 15 μg protein for each sample adjusted in 7 μl, 3 μl of 15% SDS and 10 μl of DNP solution. The mixture was kept at room temperature for 15 min, after which 7.5 μl Neutralization buffer was added. Samples (27.5 μl) were run in 10% SDS-PAGE gels, transferred into nitrocellulose (Bio-Rad, Hercules, California, USA) and blocked with 5% non-fat milk for 1h. After washing, the membrane was incubated with the first antibody (1:150) overnight at 4 °C and then for 1h with the secondary antibody (1:300) at room temperature. Membranes were incubated with ECL plus detection reagent (Bio-Rad) and scanned using Chemiluminescent scanner (Bio-Rad). Band densities were analyzed using ImageJ. Membranes were then incubated with 15% hydrogen peroxide for 30 min at room temperature and treated with actin antibody to derive a density value of actin for each lane, with which OxyBlot value was normalized.