Quantitation of protein carbonylation by dot blot

Abstract

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.

Fig. 1

Comparison of the calibration curves for the Western blot method (upper) and the dot blot method (lower). Lanes of the 14% SDS-PAGE reducing gel were loaded with varying amounts DNPH derivatized oxidized glutamine synthetase standard. From left to right, the load was 4.1, 2.0, 1.0, 0.50, 0.25, and 0.13 pmol carbonyl. The graph shows the averaged results from 2 separate blots. The regression line was fit to all points, giving y=26.6x −1.4, r²>.99. The dot blot samples were spotted in triplicate, with the top row containing no protein. All other rows were loaded with 60 ng glutamine synthetase. The carbonyl content from top to bottom was 0.13, 0.30, 0.35, 0.46, 0.57, and 0.79 pmol. The equation of the fit line is y = 16.3x + 7.7, r²>.99 with 3 measurements for each point.

Fig. 2

Comparison of the Western blot (red) and dot blot (green) methods applied to extracts exposed to varying γ irradiation. (A) Mouse liver homogenate. Four blots were averaged for the Western blot and two for the dot blot, each with triplicate analyses (B). Deinococcus radiodurans homogenate. Four blots were averaged for the Western blot, each with duplicate samples. Two blots were also averaged for the dot blot, each with sextuplicate analyses.

Fig. 3

Carbonyl content of individual Drosophila melanogaster. The average and standard deviation for triplicate analyses are shown.