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Detection of carbonylated proteins

Detection of carbonylated proteins

Detection of carbonylated proteins

All biologically relevant macromolecules, i.e. nucleic acids, membrane lipids, and proteins, are susceptible to damage by reactive oxygen species (ROS). Such oxidative damage accumulates over time during the life cycle of many organisms and has been suggested to be one possible cause of aging (Stadtman, 1992). Protein carbonylation is a widely used marker of protein oxidation (Ballesteros et al., 2001; Das et al., 2001; Mostertz and Hecker, 2003; Lounifi et al., 2013), and sensitive methods for its detection have been developed (Levine et al., 1994). It occurs by direct oxidative attack on Lys, Arg, Pro, or Thr residues of proteins, thus inhibiting or altering their activities and increasing their susceptibility toward proteolytic attack (Dunlop et al., 2002).

We have adapted published protocols for the detection of carbonylated proteins in Arabidopsis seeds (Job et al., 2005; Rajjou et al., 2008) and sunflower (Oracz et al., 2007) seeds. The appearance of carbonyl groups in oxidized proteins can be analyzed by immunodetection of 2,4-dinitrophenylhydrazone (DNP)-deriv atized protein as described in Rajjou et al. (2011) and Korolainen et al. (2002). SDS is added to the protein extract (100 µl, 10 µg/µl) to a final concentration of 0.8%. Following dialysis, four volumes of 10 mM DNPH (Sigma)/2 M HCl are added. Samples are agitated for 30 min at room temperature, and five volumes of 20/80 ice-cold TCA/acetone containing 1 mM DTT are added to each sample. The samples are centrifuged for 15 min at 15,000 g at 4°C. The precipitated protein is then washed three times with ice-cold acetone containing 1 mM DTT, then with 1 ml of 1:1 v/v ethanol:ethyl acetate, and resolubilized in the thiourea/urea lysis buffer containing 2% v/v Triton X-100 and 20 mM DTT. Proteins are separated by 1D or 2D SDS–PAGE, and transferred to nitrocellulose sheets (Bio-Rad) using standard procedures. Carbonylated proteins are revealed by incubation with rabbit anti-DNP antibodies (Chemicon, followed by incubation with antirabbit secondary antibodies conjugated to horseradish peroxidase (Sigma) and detection with the ECL kit (Roche Diagnostics) (Job et al., 2005; Rajjou et al., 2011). Relative protein carbonyl levels are quantitated by densitometric analyses of the labeled spots.
The figure (after Job et al., 2005) shows two-dimensional profiles of protein abundance and oxidation in dry mature Arabidopsis seeds. Protein stain (A) and anti-DNP immunoassay (B) are shown.


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