Dear Sir. Concerning your issue about the protocol for calculating protein carbonyl contents by spectrophotometric technique . Protein oxidation is involved in regulatory physiological events as well as in damage to tissues and is thought to play a key role in the pathophysiology of diseases and in the aging process. Protein-bound carbonyls represent a marker of global protein oxidation, as they are generated by multiple different reactive oxygen species in blood, tissues and cells. Sample preparation and stabilization are key steps in the accurate quantification of oxidation-related products and examination of physiological/pathological processes. This review therefore focuses on the sample preparation processes used in the most relevant methods to detect protein carbonyls after derivatization with 2,4-dinitrophenylhydrazine with an emphasis on measurement in plasma, cells, organ homogenates, isolated proteins and organelles. Sample preparation, derivatization conditions and protein handling are presented for the spectrophotometric and HPLC method as well as for immunoblotting and ELISA. An extensive overview covering these methods in previously published articles is given for researchers who plan to measure protein carbonyls in different samples. Or can be determined spectrophotometrically by the following: Two 1-mg aliquots are needed for each sample to be assayed. Samples are extracted in a final concentration of 10% (w/v) TCA. The precipitates are treated with 500 μL of 0.2% DNPH or 500 μL of 2 N HCl. Samples are incubated at room temperature for 1 h with vortexing at 5-min intervals. The proteins are then precipitated by adding 55 μL of 100% TCA. The pellets are centrifuged and washed three times with 500 μL of the ethanol:ethyl acetate mixture. The pellet is then dissolved in 600 μL of 6 M guanidine hydrochloride. I think the following below links may help you in your analysis:

https://chem.as.uky.edu/sites/default/files/Castegna%20et%20al%202003%20Apoptosis%20Methods%20in%20Pharm%20and%20Tox%20161-168.pdf

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4506980/pdf/main.pdf

https://www.sciencedirect.com/science/article/pii/S0003269798929323

Thanks

Thank you so much Sir (Isam Eldin Hussein Elgailani) for your kind suggestions.