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[Abstract] [Full Text] [PDF]
Post-Electrophoretic Identification of Oxidized Proteins Craig C Conrad1, John M Talent1, Christina A Malakowsky1 and Robert W Gracy 2* 1 Molecular Aging Unit, Department of Molecular Biology and
Immunology. Molecular Aging Unit, Department of Molecular
Biology and Immunology, University of North Texas Health Science Center, Fort Worth, Texas,
76107. USA. Biol. Proced. Online 2000;2:39-45. doi:10.1251/bpo17 Indexing terms: proteins; electrophoresis; oxidation-reduction. Abbreviations: DNPH, 2,4-dinitrophenylhydrazine; PVDF, polyvinyldienefluoride; DNP-antibody, dinitrophenyl-antibody; SDS, sodium dodecyl sulfate; ROS, reactive oxygen species. |
The oxidative modification of proteins has been shown to play a major role in a number of human diseases. However, the ability to identify specific proteins that are most susceptible to oxidative modifications is difficult. Separation of proteins using polyacrylamide gel electrophoresis (PAGE) offers the analytical potential for the recovery, amino acid sequencing, and identification of thousands of individual proteins from cells and tissues. We have developed a method to allow underivatized proteins to be electroblotted onto PVDF membranes before derivatization and staining. Since both the protein and oxidation proteins are quantifiable, the specific oxidation index of each protein can be determined. The optimal sequence and conditions for the staining process are (a) electrophoresis, (b) electroblotting onto PVDF membranes, (c) derivatization of carbonyls with 2,4-DNP, (d) immunostaining with anti DNP antibody, and (e) protein staining with colloidal gold. |