Protein Carbonylation Detection via OxyBlot

Protein carbonylation was detected using OxyBlot™ Protein Oxidation (Millipore S7150; Temecula, CA, USA). Samples were prepared according to the manufacturer’s protocol and an equal amount of protein (25 µg) and volume were loaded in each lane of a 12.5% polyacrylamide gel and transferred to a nitrocellulose membrane (Whatman; 10-439-196; Dassel, Germany). The transfer was confirmed using a Memcode reversible protein stain kit (Pierce Biotechnology, Fisher Scientific) and the protein was used to normalize each lane. Membranes were incubated overnight at 4 °C with primary antibody (1:150) for OxyBlot. Goat anti-rabbit horse radish peroxidase (HRP, 1:300) was the secondary antibody. Protein with 4-hydroxynonenal (4-HNE) reduced Micahel adducts were prepared as described previously [69 (link)] with the exception that 45 µg of protein was used for the gel. The membranes were developed using Amersham^TM ECLTM Western Blotting Reagents (GE Healthcare; Buckinghamshire, UK) and the results were viewed and analyzed using a Bio-Rad Chemidoc system with BioRad Chemidoc densitometry software (version 4.0.1, Catalog No. 170-9690, BioRad, Hercules, CA, USA).