Can anyone tell me what the formula to calculate the total protein carbonyl content in tissue homogenate is? Is it the same as MDA calculation using absorption coefficient value (22,000/M/Min)?
There is one review paper by Dalle-Donne et al. in one 2003 issue of Clinica Chimica Acta (volume 329) titled 'Protein carbonyl groups as biomarkers of oxidative stress'. The paper presents a variety of ways to assay for total protein carbonyls. I attached it here.
Dear Rajesh,
How do you evaluate the carbonyl content?
- If spectrophotometrically measuring the absorbance of the DNP at 360-385 nm, then use the molar absortivity of 22,000 M-1cm-1. As suggested Evangelina (manuscript Dalle-Donne) and refer the content to total protein concentration (determined for example by Lowry or Bradford method)
- If you use oxyblot, then you must to densitometer the carbonylated bands (using e.g Image J ) and then refere to total protein content (Lowry method or bradford method)
Best wishes
José Luis
Thank you friends. I had gone through the paper but i did'nt got formula. I need formula to calculate the protein carbonyl content.
I use DNP-KLH antibody with immunoblotting to determine the total carbonyl content in sperm/ tissue samples after BCA assay which i found more sensitive than spectrophotometric measurement. As @José suggested ImageJ or UnScanIt could give you relative band intensity within your sample set. If you know how much protein you have in the sample to start with any western blot formula would be helpful to start with.
Good luck
Burak
Dear Rajesh,
Using an spectrophotometrical method to analyze protein carbonyls you must be sure that exess of DNPH is completely removed from your protein pellets. Then, redisolve your protein precipitates with Guanidinium chloride 6M (in a 96 well plate, you can use 100 uL for example). After readin the absorbance at 360-380 nm, you can use a simple Lambert-beer law to calculate concentration: [carbonyls] = A/(E·l) = A/22000 (M). You can convert to nmols/mL of carbonyls because you know the added volume to your well. After that, please normalize your data to protein comtent measured previously to this assay. Your final results will be expressed as nmol Carbonyls/mg protein.
For me the spectrophotometric procedure is veri tedious and not very reproducible because of the problems to redisolve your DNPH-derivatized protein precipitates. I suggest you to use carbonyl western blot (or Oxyblot kit) for more reproducible results.
Good luck.
José Luis
Hi Rajesh,
In our Lab we use routinely a slot blot method for detection of protein carbonyl content. Please take a look on the section 2.5.2. of this link: http://www.sciencedirect.com/science/article/pii/S0006899304008157
I hope this will help you in your work.
Greetings,
Mike
We first have to determine the amount of protein in the homogenate. This is then adjusted to 4/mg of protein to keep it constant. Then we do the PC and use this same extinction coefficient. Then work back to express amount of PC/mg tissue.
i too find difficulty in calculating protein carbonyl content in plasma SAMPLES MEASURED BY ELISA -DNPH at 450 nm
I hate to get commercial or blow my own horn but I developed a kit based on published papers several years ago and did what I could to remove procedural problems to make it as easy as possible. You can read the procedure online. It's our kit K830. The main problem I found was that the protein pellet after DNP treatment was rather fragile and could easily be washed away when removing unreacted DNP so extra care needed to be used during that step. Everything else is pretty straight forward.
Dear José Luis García-Giménez ,
My friend used the calculation as per your suggestion, but she is confused regarding the volume used. I have attached her concerns.
We would greatly appreciate any response!
Thank you!!
Highly sensitive assays are available for the detection of protein carbonyls. However, in the article published by Dalle-Donne et al. (2003), derivatization with 2,4 dinitro phenyl hydrazine (DNPH) leads to the formation of a stable hydrazone, 2,4 dinitrophenyl (DNP). This article covers the process of "Protein carbonyl groups as biomarkers of oxidative stress". There are also studies by Levin et al. on the measurement process of protein carbonyls. Both articles present various ways to assay total protein carbonyls. If these papers are analyzed, they are ultimately expressed in units of nmol Carbonyl/ mg protein.
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