Search

Protocol for Notch-ligand Binding Assays Using Dynabeads   

Reviewed by
Anonymous reviewer
Download PDF How to cite Favorites Q&A Share your feedback Cited by

In this protocol

Original research article

A brief version of this protocol appeared in:
eLIFE
Apr 2017

Abstract

This protocol describes how to measure interaction between Notch receptors and their ligands by cell-based assay using Dynabeads. We have used the protocol to determine binding capacity between Notch1-transfected HEK293T cells and ligand-coated Dynabeads. Expression of Eogt in Notch1-expressing cells promoted binding toward DLL4-coated beads, but not JAG1-coated beads. The Notch-ligand assay using Dynabeads suggested that Eogt facilitates DLL4-Notch1 interaction (Sawaguchi et al., 2017).

Keywords: Notch, Ligand binding assay, Dynabeads, Dll4-Fc

Background

The Notch signal pathway regulates many types of cellular events such as proliferation, cell fate determination, and cellular differentiation in all metazoans (Mumm and Kopan, 2000). To initiate Notch signaling, extracellular domains of Notch receptors engage their ligands, Delta like (DLL) ligands or Jagged (JAG) ligands, presented on opposing cells.

Epidermal growth factor (EGF)-like domains of Notch receptors are critical for the ligand binding and modified by specific glycans including O-fucose, O-glucose, and O-GlcNAc glycans (Stanley and Okajima, 2010). Some of these glycans serve as regulators of Notch signaling pathway by modulating physical interaction between Notch receptors and ligands (Moloney et al., 2000). To investigate whether O-GlcNAc regulates Notch-ligand interaction, we developed a novel Dynabeads-based Notch-ligand binding assay. In this assay, Notch receptors expressed on HEK293T cells are incubated with Dynabeads Protein A coated with DLL4-Fc or JAG1-Fc. Unlike soluble ligands used for other binding assay, direction of Notch ligands is fixed on the beads so that they behave like ligand-expressing cells. Thus, the detected binding represents trans-binding rather than cis-binding, which occurs when Notch receptors and their ligands are expressed in the same cells. This assay demonstrated that O-GlcNAc modification of Notch1 by Eogt potentiates Notch1 binding to DLL4 without affecting JAG1 binding (Sawaguchi et al., 2017).

Copyright Sawaguchi et al. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
How to cite:  Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Sawaguchi, S., Ogawa, M. and Okajima, T. (2017). Protocol for Notch-ligand Binding Assays Using Dynabeads. Bio-protocol 7(20): e2582. DOI: 10.21769/BioProtoc.2582.
  2. Sawaguchi, S., Varshney, S., Ogawa, M., Sakaidani, Y., Yagi, H., Takeshita, K., Murohara, T., Kato, K., Sundaram, S., Stanley, P. and Okajima, T. (2017). O-GlcNAc on NOTCH1 EGF repeats regulates ligand-induced Notch signaling and vascular development in mammals. Elife 6.
Q&A

Please login to post your questions/comments. Your questions will be directed to the authors of the protocol. The authors will be requested to answer your questions at their earliest convenience. Once your questions are answered, you will be informed using the email address that you register with bio-protocol.
You are highly recommended to post your data including images for the troubleshooting.

You are highly recommended to post your data (images or even videos) for the troubleshooting. For uploading videos, you may need a Google account because Bio-protocol uses YouTube to host videos.