Protein oxidation assays

An OxyBlot Protein Oxidation Detection Kit (Sigma-Aldrich S7150) was used for immunodetection of carbonyl groups, which is a hallmark of the oxidation status of proteins introduced by oxidative reactions, following the manufacturer’s directions. Briefly, two aliquots of each specimen were used. One aliquot was used as a negative control and the other was subjected to conversion to 2,4-dinitrophenylhydrazone (DNP-hydrazone) derivative through reaction with 2,4-dinitrophenylhydrazine (DNPH). Equal amounts (20 μg) of proteins were denatured with SDS (final concentration of 6%) and treated with 1× DNPH solution (20 min) to induce derivatization. Samples designated as negative controls were treated with derivatization-control solution. After incubation, neutralization solution was added to derivatized and negative control samples. Both the treated sample and the negative control were loaded and separated by SDS-PAGE using 12% gels. Sized proteins were transferred, as described above, to a PVDF membrane, and non-specific sites were blocked by incubation with blocking/dilution buffer (1% BSA/PBS-T) for 1 h under gentle shaking. Anti-DNP antibody (1:150, Sigma-Aldrich 90451) was added for 1 h at RT. Following two washes with PBS-T, blots were incubated with NIR fluorescent secondary antibody: IRDye 800CW donkey anti-rabbit immunoglobulin (Ig) G (H + L) (Li-cor, 1:5,000 dilution). Normalization was done by total amount of protein sample per lane, using the REVERT Total Protein Stain Kit (LI-COR, P/N 926-11010). Immunostained bands were detected with the Odyssey NIR western blot detection system and quantified by densitometric scanning using the ImageStudio Lite software.