Oxyblot protein oxidation detection kit

Urinary mucosa, defined as urothelium and lamina propria, was surgically separated from the smooth muscle, homogenized using Lysing Matrix D in a FastPrep 24 instrument (MP Biomedicals, Solon, OH) following previously established protocols.^{14 (link)} Equal amounts of proteins were separated on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred to polyvinylidene fluoride membranes, which were then incubated overnight at 4°C with primary antibodies (Table 1) followed by secondary antibodies (donkey anti-rabbit horseradish peroxidase [1:5000] or sheep anti-mouse horseradish peroxidase [1:5000]; GE Amersham, Pittsburgh, PA) for 1 hour in 5% (w/v) milk TBS-T, incubated in WesternBright Quantum (Advansta, Menlo Park, CA) and then imaged on a ChemiDoc MP (Bio-Rad). As a loading control, total protein per sample was determined using Bio-Rad Stain Free SDS-PAGE gel technology (Bio-Rad). UV-activated protein fluorescence was imaged on a ChemiDoc MP (Bio-Rad). Assessment of protein carbonylation (as a marker for oxidative damage) was performed with an antibody that detects dinitrophenylhydrazine (DNPH)-derivatized carbonyl groups (OxyBlot Protein Oxidation Detection Kit, S7150; Millipore, Burlington, MA).