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Quantitative Analysis of Protein Phosphorylation Using Two-Dimensional Difference Gel Electrophoresis

  • Zhiping Deng
  • Shuolei Bu
  • Zhi-Yong Wang
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 876)

Abstract

Posttranslational modifications of proteins, especially phosphorylation and dephosphorylation, play an important role in signal transduction and cellular regulation in plants. Both 2-DE gel-based and non-gel-based proteomic technologies can monitor the changes in phosphorylation state of proteins. In this chapter, we describe two protocols for discovery and validation of differential protein phosphorylation using affinity enrichment of phosphoproteins by immobilized metal affinity chromatography (IMAC) or protein immunoprecipitation (IP) followed by two-dimensional difference gel electrophoresis (2-D DIGE). We name these methods IMAC-DIGE and IP-DIGE. For IMAC-DIGE, phosphoproteins are enriched from tissue extract using GaCl3-based IMAC and then analyzed by 2-D DIGE, which reveals changes of protein phosphorylation as protein spot shifts. IMAC enrichment improves detection of low-abundance regulatory phosphoproteins. For IP-DIGE, proteins of interest can be immunopurified and then analyzed by 2-D DIGE to confirm changes of posttranslational modifications that alter the charge or size of the proteins.

Key words

Phosphoprotein Immobilized metal affinity chromatography Proteomics 2-D DIGE Phosphorylation Posttranslational modification Brassinosteroid BZR1 

Notes

Acknowledgments

This work was supported by the Division of Chemical Sciences, Geosciences, and Biosciences, Office of Basic Energy Sciences of the U.S. Department of Energy through Grant DE-FG02-08ER15973 (to Z.-Y. W. and A. L. B.). Z. D. was partially supported by funds from Zhejiang Academy of Agricultural Sciences.

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Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  1. 1.Department of Plant BiologyCarnegie Institution for ScienceStanfordUSA
  2. 2.Institute of Virology and BiotechnologyZhejiang Academy of Agricultural ScienceHangzhouChina

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