Protein carbonyls are important compounds to measure progression of oxidative stress in tissues.
Available articles online do not have details about how to do final estimation/calculation of the carbonyl content.
You may consult the following papers to develop your protocol:
Article Direct Characterization of Protein Adducts of the Lipid Pero...
Article Oxidative damage to proteins: Spectrophotometric method for ...
Thanks Iqra, your suggestions are on point. However, I can't access the full articles.
I will be glad if you can help download and send to me as attachments
I found this method in the paper (Protein carbonyls: novel biomarkers of exposure to oxidative stress-inducing pesticides in freshwater fish Channa punctata (Bloch)), as follows:
"Protein carbonyl content was assayed by the procedure of Levine et al. (1990) as modified by Floor and Wetzel (1998). Soluble protein (0.5 mL) was reacted with 10 mM DNPH in 2 M hydrochloric acid for 1 h at room temperature and precipitated with 6% trichloroacetic acid (TCA). The pelleted protein was washed thrice by resuspension in ethanol/ethyl acetate (1:1). Proteins were then solubilized in 6 M guanidine hydrochloride, 50% formic acid and centrifuged at 16,000 × g for 5 min to remove any trace of insoluble material. The carbonyl content was measured spectrophotometrically (Shimadzu UV–vis spectrophotometer, Japan) at 366 nm. Assay was performed in triplicate and a tissue blank incubated with 2 M HCl without DNPH was included for each sample. The results were expressed as nanomoles of DNPH incorporated/mg protein based on the molar extinction coefficient of 21,000 M−1 cm−1."
Hope it helps you!
I think this research may help you:
Article Determination of protein carbonyls in plasma, cell extracts,...
Israel Ebhohimen
Have a look at the references of a review paper that summarize the recent work in this field of study, for example the spectrophotometric methods to optimize the amount of protein have been investigated in the following article
https://www.sciencedirect.com/science/article/abs/pii/S0026265X00000175
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