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In Vivo Phosphorylation: Development of Specific Antibodies to Detect the Phosphorylated PEPC Isoform for the C4 Photosynthesis in Zea mays

  • Yoshihisa UenoEmail author
  • Kumiko Yoshizawa-Kumagaye
  • Junji Emura
  • Tomoko Urabe
  • Taku Yoshiya
  • Tsuyoshi Furumoto
  • Katsura Izui
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2072)

Abstract

Phosphoenolpyruvate carboxylases (PEPCs), mostly known as the enzymes responsible for the initial CO2 fixation during C4 photosynthesis, are regulated by reversible phosphorylation in vascular plants. The phosphorylation site on a PEPC molecule is conserved not only among isoforms but also across plant species. An anti-phosphopeptide antibody is a common and powerful tool for detecting phosphorylated target proteins with high specificity. We generated two antibodies, one against a peptide containing a phosphoserine (phosphopeptide) and the other against a peptide containing a phosphoserine mimetic, (S)-2-amino-4-phosphonobutyric acid (phosphonopeptide). The amino acid sequence of the peptide was taken from the site around the phosphorylation site near the N-terminal region of the maize C4-isoform of PEPC. The former antibodies detected almost specifically the phosphorylated C4-isoform of PEPC, whereas the latter antibodies had a broader specificity for the phosphorylated PEPC in various plant species. The following procedures are described herein: (1) preparation of the phosphopeptide and phosphonopeptide; (2) preparation and purification of rabbit antibodies; (3) preparation of cell extracts from leaves for analyses of PEPC phosphorylation with antibodies; and (4) characterization of the obtained antibodies. Finally, (5) two cases involving the application of these antibodies are presented.

Key words

PEPC Phosphoenolpyruvate carboxylase C4 photosynthesis Protein phosphorylation Phosphopeptide antibody Phosphonopeptide antibody Synthetic peptide Immunodetection Zea mays Flaveria bidentis 

Notes

Acknowledgments

We thank Edanz Group (www.edanzediting.com/ac) for editing a draft of the manuscript.

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  • Yoshihisa Ueno
    • 1
    Email author
  • Kumiko Yoshizawa-Kumagaye
    • 2
  • Junji Emura
    • 2
  • Tomoko Urabe
    • 2
  • Taku Yoshiya
    • 2
  • Tsuyoshi Furumoto
    • 1
  • Katsura Izui
    • 3
  1. 1.Department of AgricultureRyukoku UniversityShigaJapan
  2. 2.Peptide Institute, Inc.OsakaJapan
  3. 3.Institute of Advanced Technology, Kindai UniversityWakayamaJapan

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