Open Access

Improving image analysis in 2DGE-based redox proteomics by labeling protein carbonyl with fluorescent hydroxylamine

  • H. Fai Poon1Email author,
  • Laila Abdullah1,
  • Jon Reed1,
  • Sarah M. Doore1,
  • Cyndi Laird1,
  • Venkat Mathura1,
  • Michael Mullan1 and
  • Fiona Crawford1
Biological Procedures Online9:91065

https://doi.org/10.1251/bpo134

Received: 31 August 2007

Accepted: 9 December 2007

Abstract

Recent advances in redox proteomics have provided significant insight into the role of oxidative modifications in cellular signalling and metabolism. At present, these techniques rely heavily on Western blots to visualize the oxidative modification and corresponding two dimensional (2D) gels for detection of total protein levels, resulting in the duplication of efforts. A major limitation associated with this methodology includes problematic matching up of gels and blots due to the differences in processing and/or image acquisition. In this study, we present a new method which allows detection of protein oxidation and total protein on the same gel to improve matching in image analysis. Furthermore, the digested protein spots are compatible with standard MALDI mass spectrometry protein identification. The methodology highlighted here may be useful in facilitating the development of biomarkers, assessing potential therapeutic targets and elucidating new mechanisms of redox signalling in redox-related conditions.

Indexing terms

electrophoresis, gel, two-dimensional oxidation-reduction protein processing, post-translational

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