Oxyblot oxidized protein detection kit

Levels of oxidized proteins in isolated samples were determined by quantifying the total protein carbonyl content following the manufacturer's instructions for the OxyBlot Oxidized Protein Detection Kit (Chemicon International, Temecula, CA, USA) (see also Supplemental Materials). Protein aggregation was determined using the filter retardation assay (Massey et al., 2006 (link)) or as material retained in the stacking gel analysis after subjecting samples to conventional SDS–PAGE (see also Supporting Information).

The Oxyblot Oxidized Protein Detection Kit was purchased from Chemicon (S7150). The procedure was according to our recent study [11 (link), 42 (link), 45 (link)]. DNPH derivatization was carried out on 6 μg of protein for 15 minutes according to manufacturer's instructions. One-dimensional electrophoresis was carried out on 12% SDS/polyacrylamide gel after DNPH derivatization. Proteins were transferred to nitrocellulose membranes, which were then incubated in the primary antibody solution (anti-DNP 1:150) for 2 hours, followed by incubation with secondary antibody solution (1:300) for one hour at room temperature. The washing procedure was repeated eight times within 40 minutes. Immunoreactive bands were visualized by enhanced chemiluminescence (ECL), which was then exposed to Biomax L film (Fuji). For quantification, ECL signals were digitized using Labwork software (UVP). On each gel, a standard control sample was loaded.

The procedure and protocol for assessing the protein expression of oxidative stress have been described in details in our previous reports [24 (link)–28 (link)]. The Oxyblot Oxidized Protein Detection Kit was purchased from Chemicon, Billerica, MA, USA (S7150). DNPH derivatization was carried out on 6 μg of protein for 15 minutes according to the manufacturer’s instructions. One-dimensional electrophoresis was carried out on 12% SDS/polyacrylamide gel after DNPH derivatization. Proteins were transferred to nitrocellulose membranes which were then incubated in the primary antibody solution (anti-DNP 1: 150) for 2 hours, followed by incubation in secondary antibody solution (1:300) for 1 hour at room temperature. The washing procedure was repeated eight times within 40 minutes. Immunoreactive bands were visualized by enhanced chemiluminescence (ECL; Amersham Biosciences, Amersham, UK) which was then exposed to Biomax L film (Kodak, Rochester, NY, USA). For quantification, ECL signals were digitized using Labwork software (UVP, Waltham, MA, USA). For oxyblot protein analysis, a standard control was loaded on each gel.