Post-electrophoretic identification of oxidized proteins
Biological Procedures Online volume 2, pages 39–45 (1999)
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.
sodium dodecyl sulfate
reactive oxygen species
Cadenas, E., Boveris, A., Ragan, C. I., and Stoppani, A. O. 1977. Archives of Biochemistry & Biophysics 248–257.
Wakeyama, H., Takeshige, K., Takayanagi, R., and Minakami, S. 1982. Biochem J 593–601.
Stadtman, E. R. 1988. J.Gerontol. B112–B120.
Davies, K. J. 1987. J.Biol.Chem. 9895–9901.
Talent, J. M., Kong, Y., and Gracy, R. W. 1998. Anal.Biochem. 31–38.
Robinson, C. E., Keshavarzian, A., Pasco, D. S., Frommel, T. O., Winship, D. H., and Holmes, E. W. 1999. Anal.Biochem. 48–57.
Bickar, D. and Reid, P. D. 1992. Anal.Biochem. 109–115.
Lim, M. J., Patton, W. F., Lopez, M. F., Spofford, K. H., Shojaee, N., and Shepro, D. 1997. Anal.Biochem. 184–195.
Neumann, H. and Mullner, S. 1998. Electrophoresis 752–757.
Rohringer, R. and Holden, D. W. 1985. Anal.Biochem. 118–127.
Mansfield, M. A. 1995. Anal.Biochem. 140–143.
Shacter, E., Williams, J. A., Lim, M., and Levine, R. L. 1994. Free Radic.Biol.Med. 429–437
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Conrad, C.C., Talent, J.M., Malakowsky, C.A. et al. Post-electrophoretic identification of oxidized proteins. Biol Proced Online 2, 39–45 (1999). https://doi.org/10.1251/bpo17