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Analysis of Protein Targets by Oxidative Stress Using the OxyBlot and Biotin–Avidin-Capture Methodology

  • Jeannette N. Stankowski
  • Simona G. Codreanu
  • Daniel C. Liebler
  • BethAnn McLaughlin
Protocol
Part of the Neuromethods book series (NM, volume 56)

Abstract

Carbonyl group formation on protein side chains is a common biochemical marker of oxidative stress and is frequently observed in a variety of acute and chronic neurological diseases including stroke, Alzheimer’s disease, and Parkinson’s disease. Given that proteins are often the immediate targets of cellular oxidative stress, it is of utmost importance to determine how adductions by reactive electrophiles and other oxidative reactions can irreversibly alter protein structure and function. Previously, protein adduction was thought to be a random process, but recently it has become increasingly clear that these protein modifications are specific and selective. In this work, two methodological approaches are presented which allow for the detection of protein carbonyl groups. While the OxyBlot methodology allows for the evaluation of general oxidative stress, the use of the novel and powerful biotin–avidin-capture methodology allows for the identification of specific proteins that have been targeted by oxidative stress.

Key words

Oxidative stress Ischemic stroke Biotin–avidin-capture methodology OxyBlot Reactive oxygen species Protein carbonyl groups Glutamate 

Notes

Acknowledgments

This work was supported by NIH grants NS050396 (BM), ES013125 (DL), and the Vanderbilt Neuroscience Predoctoral Training Fellowship T32 MH064913 (JS). Statistical and graphical support was provided by P30HD15052 (Vanderbilt Kennedy Center). The authors wish to express their gratitude to the members of the McLaughlin lab.

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Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Jeannette N. Stankowski
  • Simona G. Codreanu
  • Daniel C. Liebler
  • BethAnn McLaughlin
    • 1
  1. 1.Departments of Neurology and Pharmacology, Vanderbilt Kennedy Center for Research on Human DevelopmentVanderbilt UniversityNashvilleUSA

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