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

Eiji Kinoshita; Emiko Kinoshita-Kikuta; Tohru Koike

doi:10.1007/978-1-0716-1064-0_18

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

Methods Mol Biol. 2021:2237:217-224. doi: 10.1007/978-1-0716-1064-0_18.

Authors

Eiji Kinoshita¹, Emiko Kinoshita-Kikuta², Tohru Koike²

Affiliations

¹ Department of Functional Molecular Science, Institute of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan. kinoeiji@hiroshima-u.ac.jp.
² Department of Functional Molecular Science, Institute of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan.

PMID: 33237421
DOI: 10.1007/978-1-0716-1064-0_18

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.

Keywords: Antibody array; Biotinylated Phos-tag; Enhanced chemiluminescence; Protein phosphorylation.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Antibodies / immunology
Biotinylation
Humans
Immunoassay / methods
Luminescent Measurements / methods
Phosphoproteins / analysis*
Phosphoproteins / immunology
Polyethylene Glycols
Protein Array Analysis / methods*

Substances

Antibodies
Phosphoproteins
Polyethylene Glycols