Determination of protein carbonyl groups by immunoblotting

Free radical-mediated oxidation of proteins results in the formation of car bonyl groups in quantities that reflect the intensity of the oxidative stre ss. 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 difl uoride membrane, which was sequentially treated with 2,4-dinitrophenylhydra zine (DNPH), a primary antibody specific for the 2,4-dinitrophenol group, a nd a peroxidase-labeled second antibody. After the blots were developed wit h a chemiluminescent substrate and exposed to X-ray film, the level of immu nostaining was quantitated by densitometry, Using oxidized bovine serum alb umin as a standard and loading 5 mu g of protein per slot, the minimum dete ctable carbonyl content was approximately 60 pmol carbonyl/mg protein, When necessary, nonspecific staining by noncarbonyl constituents in complex sam ple matrices was accounted for by using sodium borohydride-treated blanks. Results by the new method were highly correlated (r = 0.932, P < 0.0001) wi th those of the standard DNPH-based spectrophotometric technique, The coeff icient of variation at a carbonyl level of 1.5 nmol/mg protein was 9.7%. Th e utility of this new method was demonstrated by measuring protein oxidatio n in cultured human colon cells (SW620) that were briefly exposed to H2O2. (C) 1999 Academic Press.