Journal
ANALYTICAL BIOCHEMISTRY
Volume 423, Issue 2, Pages 241-245Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ab.2012.01.031
Keywords
Protein carbonylation; 2,4-Dinitrophenylhydrazine; Oxidative stress; Immunoblot; Dot blot
Funding
- National Heart, Lung, and Blood Institute
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Protein carbonylation is the most commonly used measure of oxidative modification of proteins. It is frequently measured spectrophotometrically or immunochemically by derivatizing proteins with the classical carbonyl reagent, 2,4-dinitrophenylhydrazine. We developed an immunochemical dot blot method for quantitation of protein carbonylation in homogenates or purified proteins. Dimethyl sulfoxide was employed as the solvent because it very efficiently extracts proteins from tissues and keeps them soluble. It also readily dissolves 2,4-dinitrophenylhydrazine and wets polyvinylidene difluoride (PVDF) membranes. The detection limit is 0.19 +/- 0.04 pmol of carbonyl, and 60 ng of protein is sufficient to measure protein carbonyl content. This level of sensitivity allowed measurement of protein carbonylation in individual Drosophila. (C) 2012 Elsevier Inc. All rights reserved.
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