Protein carbonyl colorimetric assay

All mice were sacrificed using CO₂; next, the brain, heart, liver, and other tissues were removed with surgical instruments and placed in microcentrifuge tubes; 200 μL 0.4 M perchloric acid was added, and an ultrasonic homogenizer was used for homogenization (all procedures were performed on ice). Subsequently, the antioxidative levels in the mouse tissues were measured through superoxide dismutase (SOD) assay (Cayman Chemical Item No. 706002), glutathione peroxidase (GPx) assay (Cayman Chemical Item No. 703102), and catalase (CAT) assay (Cayman Chemical Item No. 707002). The oxidative levels were measured (and the protocol followed) through TBARS assay (Cayman Chemical Item No. 10009055), protein carbonyl colorimetric assay (Cayman Chemical Item No. 10005020), and New 8­OHdG Check ELISA (Item No. KOG-200SE, JalCA). Mitochondria DNA was extracted using a mitochondrial DNA isolation kit (Item No. K280-50, BioVision) for measuring 8-oxo-2'-deoxyguanosine (8­OHdG). All assays were conducted in accordance with the experimental standard procedures provided with the product. All oxidative and antioxidative indicators are consistent with the standard experimental test for antiaging health food identified by the Ministry of Health of the Republic of China (Taiwan).

Oxidants can react with endogenous proteins, lipids, and other materials to form oxidation products. Protein carbonylation was detected using a protein carbonyl colorimetric assay (Cayman Chemical, Ann Arbor, MI) and measured at 370 nm. The detection of lipid peroxides in the form of malondialdehyde (MDA; Cayman Chemical, Ann Arbor, MI) measured at 535 nm, and 4-Hydroxyl-2-noneal (4-HNE) and neuron specific enolase (NSE) (MyBioSource, Inc., San Diego, CA) measured at 540 nm and 450 nm, respectively. Protein nitrotyrosine concentration (StressMarq Biosciences Inc., Victoria, BC, Canada) was measured with 1:2 plasma dilution and read at 450 nm. The assays were conducted according to manufacturer’s instructions without other modifications not mentioned.

Thiobarbituric acid reactive substances (TBARS) was measured in urine and tissue homogenate of kidney cortex by adding 50 μl sample to 42 μl 0.67% thiobarbituric acid. Samples were vortexed and heated to 97°C for 60 min. After cooling the samples on ice, 50 μl methanol:1 mM NaOH (91:9) was added, the samples vortexed and centrifuged at 3000 rpm for 5 min at room temperature. The supernatant was analyzed for fluorescence using excitation/emission of 532/553 nm and the concentration calculated using a standard curve of malondialdehyde. All values were corrected for protein concentration. Concentration of TBARS in urine samples were multiplied by urine flow to get urinary TBARS excretion. Protein content was determined by DC-Protein Assay (Bio-Rad Laboratories, Hercules, CA, USA). Albuminuria was measured by rat albumin ELISA kit, (Bethyl Laboratories, Montgomery, TX, USA) according to manufacturer´s instruction. Protein carbonylation in kidney cortex was determined spectrophotometrically by protein Carbonyl Colorimetric Assay (Cayman Chemicals, MI, USA) and normalized for protein concentration.