Dear Gnanesh,

The following publication describes all available in-vitro methods for the determination of protein carbonylation:

Analysis of protein carbonylation - pitfalls and promise in commonly used methods

Rogowska-Wrzesinska, A.; Wojdyla, K.; Nedic, O.; Baron, Caroline P.; Griffith, H. R.

Published in:

Free Radical Research

Citation (APA):

Rogowska-Wrzesinska, A., Wojdyla, K., Nedic, O., Baron, C. P., & Griffith, H. R. (2014). Analysis of protein

carbonylation - pitfalls and promise in commonly used methods. Free Radical Research, 48(10), 1145-1162.

10.3109/10715762.2014.94486

Abstract

Oxidation of proteins has received a lot of attention in the last decades due to the fact that they have been shown to accumulate and to be implicated in the progression and the pathophysiology of several diseases such as Alzheimer, coronary heart diseases, etc. This has also resulted in the fact that research scientists are becoming more eager to be able to measure accurately the level of oxidized protein in biological materials, and to determine the precise site of the oxidative attack on the protein, in order to get insights into the molecular

mechanisms involved in the progression of diseases. Several methods for measuring protein carbonylation have been implemented in diff erent laboratories around the world. However, to date no methods prevail as the most accurate, reliable, and robust. The present paper aims at giving an overview of the common methods used to determine protein carbonylation in biological material as well as to highlight the limitations and the potential. The ultimate goal is to give quick tips for a rapid decision making when a method has to be selected and taking into consideration the advantage and drawback of the methods.

Keywords: carbonylation , immunoaffi nity , derivatization , mass spectrometry , standardization

To view the full review, please see attached file.

2-Another recent publication on this topic:

Redox Rep. 2009;14(4):159-66. doi: 10.1179/135100009X392601.

Protein carbonylation: avoiding pitfalls in the 2,4-dinitrophenylhydrazine assay.

Luo S1, Wehr NB.

Author information

Abstract

Protein carbonyl content is widely used as both a marker for oxidative stress and a measure of oxidative damage. Widely used methods for determination of protein carbonylation utilize the reaction of carbonyl groups with 2,4-dinitrophenylhydrazine (DNPH) to form protein-bound 2,4-dinitrophenylhydrazones. Hydrazones can be quantitated spectrophotometrically or, for greater sensitivity, detected immunochemically with anti-dinitrophenyl antibodies. Attention to methodology is important to avoid artifactual elevation in protein carbonyl measurements. We studied extracts of Escherichia coli to identify and eliminate such effects. Nucleic acid contamination caused serious artifactual increases in the protein carbonyl content determined by spectrophotometric techniques. Both in vitro synthesized DNA oligonucleotides and purified chromosomal DNA reacted strongly with 2,4-DNPH. Treatment of cell extracts with DNase+RNase or with streptomycin sulfate to precipitate nucleic acids dramatically reduced the apparent carbonyl, while exposure to proteinase K did not. The commercial kit for immunochemical detection of protein carbonylation (OxyBlot from Chemicon/Millipore) recommends a high concentration of thiol in the homogenizing buffer. We found this recommendation leads to an artifactual doubling of the protein carbonyl, perhaps due to a thiol-stimulated Fenton reaction. Avoiding oxidizing conditions, removal of nucleic acids, and prompt assay of samples can prevent artifactual effects on protein carbonyl measurements.

http://www.ncbi.nlm.nih.gov/pubmed/19695123

Hoping this will be helpful,

Rafik

thank you very much