Quantifying Oxidative Protein Damage

Samples of spinal cord, taken from six rats for each group, were homogenized in lysis buffer, as previously described (Tayebati et al., 2017 (link)). We assessed protein carbonylation by treating equal amounts of protein according to protocol of OxyBlot Protein detection kit (Millipore, USA, Cat. No. S7150). The kit provides a system to perform the immunoblot detection of carbonyl groups introduced into proteins by oxidative reactions. As a consequence, carbonyl groups are introduced into the side chains of all proteins independently of the molecular weight. The samples were separated by 8% SDS polyacrylamide gel, transferred onto nitrocellulose and blotted with the specific antibodies of the kit that recognize all the oxidized protein with different molecular weight. Band intensities were measured by densitometry with IAS 2000 image analyzer (Biosystem, Rome, Italy).