Gel electrophoretic quantitation of protein carbonyls derivatized with tritiated sodium borohydride

A method for the quantitation of protein carbonyls, which have been widely employed as markers of protein oxidative damage, is described. Protein carbonyls were derivatized with tritiated sodium borohydride and the tritiated proteins were separated on SDS-PAGE. Protein bands, visualized by Coomassie blue staining, were then excised and incubated in 30% H₂O₂ at 60°C for 48 h. Tritium, incorporated into the proteins, was quantitated by liquid scintillation counting after gel solubilization by H₂O₂. This method can be applied to the measurement of carbonylation of specific proteins as it employs SDS-PAGE and has the advantage that unreacted NaB³H₄ in the labeling reaction mixture need not be removed. The present method, when combined with immunochemical detection of protein carbonyls, should be very useful in the quantitation of oxidative damage to individual proteins.