Determination of protein carbonyl groups by immunoblotting

Free radical-mediated oxidation of proteins results in the formation of carbonyl groups in quantities that reflect the intensity of the oxidative stress. We have developed an immunochemical technique for the quantification of carbonyl groups in protein samples prepared from small tissue samples and cell cultures. Protein samples were slot-blotted onto a polyvinylidene difluoride membrane, which was sequentially treated with 2,4-dinitrophenylhydrazine (DNPH), a primary antibody specific for the 2,4-dinitrophenol group, and a peroxidase-labeled second antibody. After the blots were developed with a chemiluminescent substrate and exposed to X-ray film, the level of immunostaining was quantitated by densitometry. Using oxidized bovine serum albumin as a standard and loading 5 microg of protein per slot, the minimum detectable carbonyl content was approximately 60 pmol carbonyl/mg protein. When necessary, nonspecific staining by noncarbonyl constituents in complex sample matrices was accounted for by using sodium borohydride-treated blanks. Results by the new method were highly correlated (r = 0.932, P < 0.0001) with those of the standard DNPH-based spectrophotometric technique. The coefficient of variation at a carbonyl level of 1.5 nmol/mg protein was 9.7%. The utility of this new method was demonstrated by measuring protein oxidation in cultured human colon cells (SW620) that were briefly exposed to H2O2.