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Neutral Phosphate-Affinity SDS-PAGE System for Profiling of Protein Phosphorylation

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Proteomic Profiling

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1295))

Abstract

In this chapter, we describe a standard protocol for phosphate-affinity SDS-PAGE that uses a dizinc(II) complex of the phosphate-binding molecule Phos-tag in conjunction with a neutral-pH gel system (Zn2+–Phos-tag SDS-PAGE) to detect shifts in the mobilities of phosphoproteins. A previous protocol for affinity electrophoresis that uses polyacrylamide-bound Mn2+-Phos-tag and Laemmli’s buffer system under conditions of alkaline pH has limitations in separating certain phosphoproteins. The current protocol provides major improvements in separation and detection of various phosphorylated protein species. We here introduce two neutral-pH gel systems buffered with Bis–Tris–HCl and Tris–AcOH, respectively, for Zn2+–Phos-tag SDS-PAGE, and we also discuss their characteristics on the basis of comparative studies on phosphorylation profiling of proteins with a wide range of molecular masses. Each analytical procedure, from the beginning of gel preparation to the end of electrophoresis, requires 2.5–5 h with either buffer system.

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Acknowledgments

This work was supported in part by KAKENHI Grants (24590050, 25293005, 25560417, 25117718, and 26460036) and a research grant from the Takeda Science Foundation.

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Authors and Affiliations

  1. Department of Functional Molecular Science, Institute of Biomedical & Health Sciences, Hiroshima University, Hiroshima, 734-8553, Japan

    Emiko Kinoshita-Kikuta, Eiji Kinoshita & Tohru Koike

  2. Department of Functional Molecular Science, Graduate School of Biomedical Sciences, Hiroshima University, Kasumi 1-2-3, Minami-ku, Hiroshima, 734-8553, Japan

    Eiji Kinoshita

Authors
  1. Emiko Kinoshita-Kikuta
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  2. Eiji Kinoshita
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  3. Tohru Koike
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Corresponding author

Correspondence to Eiji Kinoshita .

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Editors and Affiliations

  1. Bio-Rad Laboratories GmbH, Munich, Germany

    Anton Posch

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Kinoshita-Kikuta, E., Kinoshita, E., Koike, T. (2015). Neutral Phosphate-Affinity SDS-PAGE System for Profiling of Protein Phosphorylation. In: Posch, A. (eds) Proteomic Profiling. Methods in Molecular Biology, vol 1295. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2550-6_24

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  • DOI: https://doi.org/10.1007/978-1-4939-2550-6_24

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-2549-0

  • Online ISBN: 978-1-4939-2550-6

  • eBook Packages: Springer Protocols

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