Oxyblot protein oxidation

Carbonylated proteins were analyzed using the OxyBlot kit according to the manufacturer’s instructions: OxyBlotTM Protein Oxidation (Merck Millipore, Darmstadt, Germany). Briefly, samples (15 µg protein) were denatured by adding 5 μL of 12% SDS to a final concentration of 6% SDS. The samples were derivatized by adding 10 μL of 1X DNPH (2,4-dinitrophenolhydrazine) solution and incubated (at RT for 15 min). Samples were neutralized (7.5 μL of neutralization solution) and the derivatized proteins were separated by SDS/PAGE (see above). The primary antibody used was against DNPH and detected by luminescence (SuperSignal detection system kit: Pierce Chemical, Dallas, TX, USA). The bands were quantified (Gel-Logic 2200-PRO Bioscience, London, UK), and analyzed (Molecular Imaging software).

Oxidized proteins were analyzed on tissues of 5 mice for each group by means of the OxyBlotTM Protein Oxidation (Merck Millipore, Darmstadt, Germany), conceding to the manufacturer’s instructions. Briefly, 15 μg protein of each sample were denatured by adding 5 μL of 12% SDS (Sigma-Aldrich, St. Louis, MO, USA). Samples were derivatized by adding 10 μL of a 1 × 2,4 dinitrophenolhydrazine (DNPH, Sigma-Aldrich, St. Louis, MO, USA) solution and incubated for 15 min at RT. Subsequently, samples were neutralized with 7.5 μL of a neutralization solution and the proteins were separated by SDS/PAGE. The primary antibody used was against DNPH and revealed by luminescence (SuperSignal detection system kit: Pierce Chemical, Dallas, TX, USA). The bands were quantified, normalizing the pixels in each lane to the loading control band (Gel-Logic 2200-PRO Bioscience, London, UK), and analyzed (Molecular Imaging software).