Oxidative Stress Assessment in Muscle and Liver

Oxidative stress in both skeletal muscle and liver was examined using a glutathione assay kit from Cayman Chemical (Ann Arbor, MI) according to the manufacturer’s instructions. Oxidative protein carbonylation assays in both skeletal muscle and liver were performed following Western blot by using an OxyBlot Protein Detection Kit from Millipore (Billerica, MA) according to the manufacturer’s instructions. The carbonyl groups in protein side chains were derivatized to DNP-hydrazone by reaction with DNPH following the manufacturer’s instructions. After the derivatization of the protein sample, 1-dimensional electrophoresis was carried out on a 10% SDS-PAGE gel. Proteins were transferred to PVDF membranes. After incubation with anti-DNP antibody, the blot was developed using a chemiluminescence detection system. ATP levels were analyzed in liver and skeletal muscle extracts as described previously [14] (link). ATP concentrations were determined using the luciferase-based ATP-assay from Roche (Mannheim, Germany), and values were normalized to mtDNA content.