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High Sensitive Quantitative Binding Assays Using a Nanoluciferase-Fused Probe for Analysis of ALG-2-Interacting Proteins

  • Wei Zhang
  • Rina Matsuo
  • Terunao Takahara
  • Hideki Shibata
  • Masatoshi MakiEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1929)

Abstract

Many non-catalytic cellular proteins exert biological functions by formation of stable or transient complexes with other proteins. Analysis of the signal-induced physical interactions is important to understand their physiological roles in cells. Here we describe a biochemical method for assessing the binding of ALG-2 (gene name, PDCD6) to its target proteins that are immunoprecipitated from cell lysates. Application of nanoluciferase (Nluc)-fused ALG-2 enables a rapid quantitative evaluation of Ca2+-dependent interactions of target proteins with ALG-2 in vitro binding assays.

Key words

Protein–protein interaction ALG-2 Calcium-binding protein EF-hand NanoLuc In vitro binding 

Notes

Acknowledgments

This work was supported by JSPS KAKENHI Grant Number 17H03803 (Grant-in-Aid for Scientific Research (B) to M.M.). We thank current and past members of the Laboratory of Molecular and Cellular Regulation for valuable suggestions and discussion. Wei Zhang and Rina Matsuo contributed equally to this work.

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Wei Zhang
    • 1
  • Rina Matsuo
    • 1
  • Terunao Takahara
    • 1
  • Hideki Shibata
    • 1
  • Masatoshi Maki
    • 1
    Email author
  1. 1.Department of Applied Molecular BiosciencesGraduate School of Bioagricultural Sciences, Nagoya UniversityNagoyaJapan

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