Abstract
Protein phosphorylation plays critical roles in multiple aspects of cellular events. Site- and phosphorylation state-specific antibodies are indispensable to analyze spatially and temporally distribution of protein phosphorylation in cells. Such information provides some clues of its biological function. Here, we describe a strategy to design a phosphopeptide as an antigen for a site- and phosphorylation state-specific antibody. Importantly, this strategy is also applicable to the production of other types of antibodies, which specifically recognize the site-specific modification, such as acetylation, methylation, and proteolysis. This protocol also focuses on the screening for monoclonal version of a site- and phosphorylation state-specific antibody.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Graves JD, Krebs EG (1999) Protein phosphorylation and signal transduction. Pharmacol Ther 82(2–3):111–121
Nishizuka Y (1992) Intracellular signaling by hydrolysis of phospholipids and activation of protein kinase C. Science 258(5082):607–614
Nurse P (2000) A long twentieth century of the cell cycle and beyond. Cell 100(1):71–78
Zhou BB, Elledge SJ (2000) The DNA damage response: putting checkpoints in perspective. Nature 408(6811):433–439
Bishop AL, Hall A (2000) Rho GTPases and their effector proteins. Biochem J 348(Pt 2):241–255
Boyle WJ, van der Geer P, Hunter T (1991) Phosphopeptide mapping and phosphoamino acid analysis by two-dimensional separation on thin-layer cellulose plates. Methods Enzymol 201:110–149
Sternberger LA, Sternberger NH (1983) Monoclonal antibodies distinguish phosphorylated and nonphosphorylated forms of neurofilaments in situ. Proc Natl Acad Sci U S A 80(19):6126–6130
Nishizawa K, Yano T, Shibata M, Ando S, Saga S, Takahashi T, Inagaki M (1991) Specific localization of phosphointermediate filament protein in the constricted area of dividing cells. J Biol Chem 266(5):3074–3079
Yano T, Taura C, Shibata M, Hirono Y, Ando S, Kusubata M, Takahashi T, Inagaki M (1991) 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(3):1144–1151
Matsuoka Y, Nishizawa K, Yano T, Shibata M, Ando S, Takahashi T, Inagaki M (1992) Two different protein kinases act on a different time schedule as glial filament kinases during mitosis. EMBO J 11(8):2895–2902
Nagata K, Izawa I, Inagaki M (2001) A decade of site- and phosphorylation state-specific antibodies: recent advances in studies of spatiotemporal protein phosphorylation. Genes Cells 6(8):653–664
Izawa I, Inagaki M (2006) Regulatory mechanisms and functions of intermediate filaments: a study using site- and phosphorylation state-specific antibodies. Cancer Sci 97(3):167–174. doi:10.1111/j.1349-7006.2006.00161.x
Goto H, Inagaki M (2007) Production of a site- and phosphorylation state-specific antibody. Nat Protoc 2(10):2574–2581. doi:10.1038/nprot.2007.374
Czernik AJ, Girault JA, Nairn AC, Chen J, Snyder G, Kebabian J, Greengard P (1991) Production of phosphorylation state-specific antibodies. Methods Enzymol 201:264–283
White DA, Belyaev ND, Turner BM (1999) Preparation of site-specific antibodies to acetylated histones. Methods San Diego, Calif 19(3):417–424
Perez-Burgos L, Peters AH, Opravil S, Kauer M, Mechtler K, Jenuwein T (2004) Generation and characterization of methyl-lysine histone antibodies. Methods Enzymol 376:234–254
Howell BJ, McEwen BF, Canman JC, Hoffman DB, Farrar EM, Rieder CL, Salmon ED (2001) Cytoplasmic dynein/dynactin drives kinetochore protein transport to the spindle poles and has a role in mitotic spindle checkpoint inactivation. J Cell Biol 155(7):1159–1172. doi:10.1083/jcb.200105093
Kasahara K, Goto H, Enomoto M, Tomono Y, Kiyono T, Inagaki M (2010) 14-3-3gamma mediates Cdc25A proteolysis to block premature mitotic entry after DNA damage. EMBO J 29(16):2802–2812. doi: 10.1038/emboj. 2010.157, emboj 2010157 [pii]
Hayashi K, Yonemura S, Matsui T, Tsukita S (1999) Immunofluorescence detection of ezrin/radixin/moesin (ERM) proteins with their carboxyl-terminal threonine phosphorylated in cultured cells and tissues. J Cell Sci 112(Pt 8):1149–1158
Acknowledgements
This work was supported in part by Grants-in-Aid for Scientific Research on Innovative 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, Japan; by a Grant-in-aid for the Third Term Comprehensive 10-Year Strategy for Cancer Control from the Ministry of Health, Labour and Welfare, Japan; by the Uehara Memorial Foundation; by the Astellas Foundation for Research on Metabolic Disorders; by the Naito Foundation; by the Daiichi-Sankyo Foundation of Life Science; and by the Takeda Science Foundation.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2014 Springer Science+Business Media, New York
About this protocol
Cite this protocol
Goto, H., Inagaki, M. (2014). Method for the Generation of Antibodies Specific for Site and Posttranslational Modifications. In: Ossipow, V., Fischer, N. (eds) Monoclonal Antibodies. Methods in Molecular Biology, vol 1131. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-992-5_2
Download citation
DOI: https://doi.org/10.1007/978-1-62703-992-5_2
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-991-8
Online ISBN: 978-1-62703-992-5
eBook Packages: Springer Protocols