Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

Production of a site- and phosphorylation state-specific antibody

Nature Protocols volume 2, pages 2574–2581 (2007)Cite this article

Abstract

Protein phosphorylation plays important roles in various aspects of cellular events. Visualization of site-specific phosphorylation in cells is of great importance not only to analyze spatial and temporal distribution but also to investigate biological function. Now, site- and phosphorylation state-specific antibodies are widely utilized as the most powerful tools for these analyses. This protocol details a method to produce the polyclonal version of such an antibody by immunizing a synthetic phosphopeptide corresponding to a protein phosphorylated at targeted site(s). This protocol is also applicable to the production of other types of antibodies, which specifically recognize the site-specific modification, such as acetylation, methylation and proteolysis. The protocol can be completed in 2–3 months.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Design of synthetic peptides for producing each site- and phosphorylation state-specific antibody.
Figure 2: Schematic diagram showing the emulsification of adjuvant/antigen and the affinity-purification of antibodies.
Figure 3: Purification of a site- and phosphorylation state-specific antibody.
Figure 4: Characterization and utilization of a site- and phosphorylation state-specific antibody (AK8578)14 against phosphoSer857 and phosphoSer858 on inner centromere protein (INCENP).
Figure 5: Characterization and utilization of a site- and phosphorylation state-specific antibody (αPH10)16 against phosphoSer10 on histone H3.

Similar content being viewed by others

References

  1. Graves, J.D. & Krebs, E.G. Protein phosphorylation and signal transduction. Pharmacol. Ther. 82, 111–121 (1999).

    Article  CAS  PubMed  Google Scholar 

  2. Nishizuka, Y. Intracellular signaling by hydrolysis of phospholipids and activation of protein kinase C. Science 258, 607–614 (1992).

    Article  CAS  PubMed  Google Scholar 

  3. Nurse, P. A long twentieth century of the cell cycle and beyond. Cell 100, 71–78 (2000).

    Article  CAS  PubMed  Google Scholar 

  4. Zhou, B.B. & Elledge, S.J. The DNA damage response: putting checkpoints in perspective. Nature 408, 433–439 (2000).

    Article  CAS  PubMed  Google Scholar 

  5. Bishop, A.L. & Hall, A. Rho GTPases and their effector proteins. Biochem. J. 348, 241–255 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Boyle, W.J., van der Geer, P. & Hunter, T. Phosphopeptide mapping and phosphoamino acid analysis by two-dimensional separation on thin-layer cellulose plates. Methods Enzymol. 201, 110–149 (1991).

    Article  CAS  PubMed  Google Scholar 

  7. Sternberger, L.A. & Sternberger, N.H. Monoclonal antibodies distinguish phosphorylated and nonphosphorylated forms of neurofilaments in situ. Proc. Natl. Acad. Sci. USA 80, 6126–6130 (1983).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Nishizawa, K. et al. Specific localization of phosphointermediate filament protein in the constricted area of dividing cells. J. Biol. Chem. 266, 3074–3079 (1991).

    CAS  PubMed  Google Scholar 

  9. Yano, T. et al. A monoclonal antibody to the phosphorylated form of glial fibrillary acidic protein: application to a non-radioactive method for measuring protein kinase activities. Biochem. Biophys. Res. Commun. 175, 1144–1151 (1991).

    Article  CAS  PubMed  Google Scholar 

  10. Nagata, K., Izawa, I. & Inagaki, M. A decade of site- and phosphorylation state-specific antibodies: recent advances in studies of spatiotemporal protein phosphorylation. Genes Cells 6, 653–664 (2001).

    Article  CAS  PubMed  Google Scholar 

  11. Czernik, A.J. et al. Production of phosphorylation state-specific antibodies. Methods Enzymol. 201, 264–283 (1991).

    Article  CAS  PubMed  Google Scholar 

  12. White, D.A., Belyaev, N.D. & Turner, B.M. Preparation of site-specific antibodies to acetylated histones. Methods 19, 417–424 (1999).

    Article  CAS  PubMed  Google Scholar 

  13. Perez-Burgos, L. et al. Generation and characterization of methyl-lysine histone antibodies. Methods Enzymol. 376, 234–254 (2004).

    Article  CAS  PubMed  Google Scholar 

  14. Goto, H. et al. Complex formation of Plk1 and INCENP required for metaphase-anaphase transition. Nat. Cell Biol. 8, 180–187 (2006).

    Article  CAS  PubMed  Google Scholar 

  15. Carmena, M. & Earnshaw, W.C. The cellular geography of aurora kinases. Nat. Rev. Mol. Cell Biol. 4, 842–854 (2003).

    Article  CAS  PubMed  Google Scholar 

  16. Goto, H. et al. Identification of a novel phosphorylation site on histone H3 coupled with mitotic chromosome condensation. J. Biol. Chem. 274, 25543–25549 (1999).

    Article  CAS  PubMed  Google Scholar 

  17. Hendzel, M.J. et al. Mitosis-specific phosphorylation of histone H3 initiates primarily within pericentromeric heterochromatin during G2 and spreads in an ordered fashion coincident with mitotic chromosome condensation. Chromosoma 106, 348–360 (1997).

    Article  CAS  PubMed  Google Scholar 

  18. Kawajiri, A. & Inagaki, M. Approaches to study phosphorylation of intermediate filament proteins using site-specific and phosphorylation state-specific antibodies. Methods Cell Biol. 78, 353–371 (2004).

    Article  CAS  PubMed  Google Scholar 

  19. Bishop, J.D. & Schumacher, J.M. Phosphorylation of the carboxyl terminus of inner centromere protein (INCENP) by the Aurora B kinase stimulates Aurora B kinase activity. J. Biol. Chem. 277, 27577–27580 (2002).

    Article  CAS  PubMed  Google Scholar 

  20. Honda, R., Körner, R. & Nigg, E.A. Exploring the functional interactions between Aurora B, INCENP, and survivin in mitosis. Mol. Biol. Cell 14, 3325–3341 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Hsu, J.Y. et al. Mitotic phosphorylation of histone H3 is governed by Ipl1/aurora kinase and Glc7/PP1 phosphatase in budding yeast and nematodes. Cell 102, 279–291 (2000).

    Article  CAS  PubMed  Google Scholar 

  22. Goto, H., Yasui, Y., Nigg, E.A. & Inagaki, M. Aurora-B phosphorylates histone H3 at serine28 with regard to the mitotic chromosome condensation. Genes Cells 7, 11–17 (2002).

    Article  CAS  PubMed  Google Scholar 

  23. Tsujimura, K. et al. Visualization and function of vimentin phosphorylation by cdc2 kinase during mitosis. J. Biol. Chem. 269, 31097–31106 (1994).

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This work was supported in part by Grants-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Culture, Sports, Science and Technology, Japan, by Grants-in-Aid for Scientific Research from Japan Society for the Promotion of Science, by a Grant-in-aid for the Third Term Comprehensive 10-Year Strategy for Cancer Control from the Ministry of Health and Welfare, Japan, by Astellas Foundation for Research on Metabolic Disorders, by Aichi Cancer Research Foundation, by The Ichiro Kanehara Foundation and by The Mochida Memorial Foundation for Medical and Pharmaceutical Research. Protocol derived from http://dx.doi.org/10.1038/nprot.2007.31.

Author information

Authors and Affiliations

  1. Division of Biochemistry, Aichi Cancer Center Research Institute, Nagoya, 464-8681, Aichi, Japan

    Hidemasa Goto & Masaki Inagaki

  2. Department of Cellular Oncology, Nagoya University Graduate School of Medicine, Nagoya, 466-8550, Aichi, Japan

    Masaki Inagaki

Authors
  1. Hidemasa Goto
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Masaki Inagaki
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Masaki Inagaki.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Goto, H., Inagaki, M. Production of a site- and phosphorylation state-specific antibody. Nat Protoc 2, 2574–2581 (2007). https://doi.org/10.1038/nprot.2007.374

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2007.374

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing