I need a hypothetical example as an answer for my question for a better understanding. After getting the absorbance values of the protein samples by DNPH method, how can i express my results in units of nmol/mg protein from these absorbance values? the assay is protein carbonylation assay
First of all, the assay you were based on has some dilutions which must be taken into account.
To continue , you have two measurements [one blank (A blank) and one of the sample Asample)]. Subtract A blank from A sample. Then you have to multiply with the molecular extinction coefficient of DNPH which is 0.022. After that you have to multiply with a dilution coefficient which depends on your technique. For instance if the sample had a volume of 50 μL and the cuvette accepted a volume of 1000 μL in order to measure absorbance, your dilution coefficient is 1000/20 = 50. Then ypu have to multiply with other dilution coefficients depending on: a) how many times you diluted the sample during homogenization and b) during protein precipitation (in case you used TCA or something else). After all these mathematics you receive a result in nmol/mL
For the protein estimation I usually follow the Bradford method in intact aliquots (without having started protein carbonyls assay). After I take the measurements of the protein assay (mg/mL) I put these notes aside until the carbonyl assay is completed.
After you calculate the two numbers (protein carbonyls and protein concentration) the first is divided by the second and you have your result which is expressed in nmol/mg protein.
Example: your protein carbonyls result is 280 nmol/mL and the protein concentration after the Bradford method is 158.025 mg/mL. Your final result is 280/158.025= 1.7719 nmol/ mg protein
Ioannis,
Your assuming that the number of carbonyls is proportional to the mass and the molecular weight of the protein or peptide. This is not true. While all amino acids contain at least 1 carbonyl group the molecular weight of the 20 physiological amino acids can vary. What is your carbonyl assay (titration, FTIR...)?
No, I am not assuming that. Besides the expression in ng/ mg protein corrects all possible errors. Our method is based on those of
Patsoukis, N., Zervoudakis, G., Panagopoulos, N. T., Georgiou, C. D., Angelatou, F., & Matsokis, N. A. (2004). Thiol redox state (TRS) and oxidative stress in the mouse hippocampus after pentylenetetrazol-induced epileptic seizure. Neuroscience letters, 357(2), 83-86.
Also, a review of this technique can be seen on the review of
Veskoukis, A. S., Kyparos, A., Paschalis, V., & Nikolaidis, M. G. (2016). Spectrophotometric assays for measuring redox biomarkers in blood. Biomarkers, 21(3), 208-217.
who approve of this method.
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