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Detection of Heterodimerization of Protein Isoforms Using an in Situ Proximity Ligation Assay

Detection of Heterodimerization of Protein Isoforms Using an in Situ Proximity Ligation Assay

Published on: October 20, 2018

In-Solution SH2 Domain Binding Assay Based on Proximity Ligation.

Kazuya Machida¹

¹Raymond and Beverly Sackler Laboratory of Genetics and Molecular Medicine, Department of Genetics and Genome Sciences, University of Connecticut School of Medicine, 400 Farmington Avenue, Farmington, CT, 06032, USA. machida@uchc.edu.

Methods in Molecular Biology (Clifton, N.J.)

|January 17, 2017

Summary

This summary is machine-generated.

We developed SH2-PLA, a novel assay for studying protein interactions in tyrosine kinase signaling. This high-throughput method uses proximity ligation and real-time PCR for rapid and sensitive analysis of SH2 domain binding.

Keywords:

EGFR Grb2 In-solution binding assay Proximity ligation Real-time PCR SH2 domain

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Area of Science:

Biochemistry
Molecular Biology
Cell Signaling

Background:

Protein-protein interactions mediated by SH2 domains are crucial for tyrosine kinase pathway specificity.
Existing methods like far-Western and pull-down assays for assessing these interactions are low-throughput.
There is a need for rapid and sensitive assays to study these interactions in cell signaling.

Purpose of the Study:

To develop a novel, high-throughput assay for assessing SH2 domain binding to target proteins.
To leverage proximity ligation and real-time PCR for enhanced speed and sensitivity.
To provide a valuable tool for studying tyrosine kinase signaling pathways.

Main Methods:

Development of SH2-PLA, an in-solution assay.
Utilizing proximity ligation for sensitive detection of binding events.
Employing real-time PCR for quantitative analysis.
Assaying SH2 domain binding using small volumes of cell lysate.

Main Results:

SH2-PLA enables rapid assessment of SH2 domain binding to target proteins.
The assay demonstrates high sensitivity and requires minimal sample input.
SH2-PLA offers a significant improvement in throughput compared to traditional methods.

Conclusions:

SH2-PLA is an effective and efficient new tool for studying SH2 domain-mediated protein interactions.
This assay facilitates the study of tyrosine kinase signaling.
The method's speed and sensitivity make it attractive for research in cell signaling and related fields.