Protein carbonyl content colorimetric assay kit

The test compound, i.e., BC, was obtained from palm oil mill effluent (supplied by Palm Oil Mill Sdn. Bhd., Penang, Malaysia). The reference control drug, i.e., CoQ10, was procured from Pharma Nord Sdn Bhd, Kuala Lumpur, Malaysia. 5,5-dithibis(2-nitrobenzoic acid) (DTNB), ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), 10 mM of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), β-cryptoxanthin, nicotinamide adenine dinucleotide phosphate (NADPH), nicotinamide adenine dinucleotide hydrogen (NADH), protein carbonyl content colorimetric assay kit, and ATP were purchased from Merck Sdn Bhd, Selangor, Malaysia. The SOD2 enzyme-linked immunosorbent assay (ELISA) kit was procured from LSBio, Selangor, Malaysia. Blood urea nitrogen (BUN) and creatinine kits were procured from Biogenix Inc. Pvt. Ltd., Lucknow, India. protein carbonyl content colorimetric assay kit was procured from Sigma-Aldrich Sdn. Bhd., Petaling Jaya, Malaysia. The mitochondrial complex I activity colorimetric assay kit was procured from Abcam Sdn. Bhd., Kuala Lumpur, Malaysia. All the chemical reagents were used as an analytical grade.

The renal mitochondrial carbonylated protein content was estimated as described in the protein carbonyl content colorimetric assay kit (Sigma-Aldrich (M) Sdn. Bhd., Petaling Jaya, Malaysia). Briefly, 100 μL of renal mitochondrial aliquot was mixed with 100 μL of 2,4-dinitrophenylhydrazine (DNPH) and vortexed followed by incubation at room temperature (37 °C) for 10 min. Then, 30 μL of trichloroacetic acid was mixed in each test tube sample. Crucially, 10 μL of streptozocin and 200 μL of guanidine solution were added and sonicated to resolubilization of proteins. All the samples were subjected to the formation of stable chromogen, i.e., dinitrophenyl (DNP) hydrazine. The absorbance changes of colored chromogen were estimated at 375 nm by using a UV-1800 Shimadzu Spectrophotometer (Shimadzu Corporation, Kyoto, Japan). The standard plot was prepared with 1 to 15 nanomoles of carbonylated protein. The value of renal mitochondrial carbonylated protein content was expressed as nM/mg of protein.