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Determining Protein Phosphorylation Status Using Antibody Arrays and Phos-Tag Biotin

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Antibody Arrays

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2237))

Abstract

We describe here a standard protocol for determining the phosphorylation status of protein multiplexes using antibody arrays and a biotinylated Phos-tag with a dodeca(ethylene glycol) spacer (Phos-tag Biotin). The procedure is based on an antibody microarray technique used in conjunction with an enhanced chemiluminescence system, and it permits the simultaneous and highly sensitive detection of multiple phosphoproteins in a cell lysate. By using this procedure, we have demonstrated the quantitative detection of the entire phosphorylation status of a target protein involved in intracellular signaling.

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Acknowledgments

This work was supported in part by KAKENHI Grants 19K07147 to E.K., 18K06596 to E.K.-K., and 17K08237 to T.K.

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Authors and Affiliations

  1. Department of Functional Molecular Science, Institute of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan

    Eiji Kinoshita, Emiko Kinoshita-Kikuta & Tohru Koike

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

Correspondence to Eiji Kinoshita .

Editor information

Editors and Affiliations

  1. RayBiotech, Peachtree Corners, GA, USA

    Kelly C. Whittaker

  2. RayBiotech Life, Peachtree Corners, GA, USA

    Ruo-Pan Huang

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Kinoshita, E., Kinoshita-Kikuta, E., Koike, T. (2021). Determining Protein Phosphorylation Status Using Antibody Arrays and Phos-Tag Biotin. In: Whittaker, K.C., Huang, RP. (eds) Antibody Arrays. Methods in Molecular Biology, vol 2237. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1064-0_18

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  • DOI: https://doi.org/10.1007/978-1-0716-1064-0_18

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1063-3

  • Online ISBN: 978-1-0716-1064-0

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