Post-electrophoretic identification of oxidized proteins |
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Authors: | Craig C Conrad John M Talent Christina A Malakowsky Robert W Gracy PhD |
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Institution: | (1) Molecular Aging Unit, Department of Molecular Biology and Immunology, University of North Texas Health Science Center, 76107 Fort Worth, Texas;(2) Office of Research and Biotechnology, ME-2 Room 806, 3500 Camp Bowie Blvd, 76107 Fort Worth, Texas |
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Abstract: | 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. |
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