Quantification of Protein Interaction Network Dynamics using Multiplexed Co-Immunoprecipitation

Emily  A. Brown; Steven  C. Neier; Claudia Neuhauser; Adam  G. Schrum; Stephen  E.P. Smith

doi:10.3791/60029

Quantifizierung der Protein-Interaktionsnetzwerkdynamik mit Multiplex-Co-Immunopräzipitation

JoVE Journal
Biochemistry

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JoVE Journal Biochemistry

Quantification of Protein Interaction Network Dynamics using Multiplexed Co-Immunoprecipitation

Quantifizierung der Protein-Interaktionsnetzwerkdynamik mit Multiplex-Co-Immunopräzipitation

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07:57 min

August 21, 2019

DOI: 10.3791/60029-v

Emily A. Brown^1,2, Steven C. Neier^3,4, Claudia Neuhauser⁵, Adam G. Schrum^6,7,8, Stephen E.P. Smith^1,2,9

¹Center for Integrative Brain Research,Seattle Children's Research Institute, ²Graduate Program in Neuroscience,University of Washington, ³Department of Cancer Immunology and Virology, Dana-Farber Cancer Institute, Department of Medicine,Harvard Medical School, ⁴Broad Institute of Harvard and MIT, ⁵Department of Mathematics,University of Houston, ⁶Department of Molecular Microbiology and Immunology, School of Medicine,University of Missouri, ⁷Department of Surgery, School of Medicine,University of Missouri, ⁸Department Bioengineering, College of Engineering,University of Missouri, ⁹Department of Pediatrics,University of Washington

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Overview

Quantitative multiplexed co-immunoprecipitation (QMI) allows for the simultaneous measurement of numerous protein-protein interactions within a defined network. This method is efficient, requiring minimal biomaterial and no genetically engineered tags.

Key Study Components

Area of Science

Neuroscience
Biochemistry
Cell Biology

Background

QMI enables the detection of differences in protein interactions between samples.
The assay can be adapted for various protein interaction networks.
It allows for the collection of network-scale data about signal transduction systems.
Careful pipetting and setup are crucial to avoid errors.

Purpose of Study

To measure binary interactions in protein networks.
To analyze how co-associations change in a coordinated manner.
To develop a reliable assay for studying complex biological systems.

Methods Used

Simultaneous co-immunoprecipitation in 96 well plates.
Lysis of samples using suitable detergents with inhibitors.
Bioinformatics techniques for data analysis.
Overnight assays for data collection.

Main Results

QMI provides sensitive detection of protein interactions.
Facilitates the study of complex signaling pathways.
Enables high-throughput analysis of protein networks.
Demonstrates the effectiveness of multiplexing in immunoprecipitation.

Conclusions

QMI is a powerful tool for studying protein interactions.
It simplifies the process of analyzing complex biological data.
Future applications may expand to various fields of research.

Frequently Asked Questions

What is QMI?

QMI stands for Quantitative Multiplex Immunoprecipitation, a method for detecting protein interactions.

How does QMI work?

It uses flow cytometry to measure differences in protein interactions between samples.

What are the advantages of QMI?

QMI requires less biomaterial and does not need genetically engineered tags.

What is the main application of QMI?

It is used to study protein-protein interactions in various biological systems.

How long does it take to develop a QMI assay?

The assay is time-consuming to develop, but data can be collected overnight once established.

What precautions should be taken during QMI?

Accurate pipetting and careful setup are crucial to prevent errors.

Quantitative Multiplex Immunoprecipitation (QMI) verwendet Durchflusszytometrie zum empfindlichen Nachweis von Unterschieden in der Häufigkeit gezielter Protein-Protein-Wechselwirkungen zwischen zwei Proben. QMI kann mit einer kleinen Menge an Biomaterial durchgeführt werden, erfordert keine gentechnisch veränderten Tags und kann für jedes zuvor definierte Protein-Interaktionsnetzwerk angepasst werden.

Quantitative Multiplex-Co-Immunpräzipation (QMI) ermöglicht es Benutzern, gleichzeitig Hunderte von binären Wechselwirkungen in einem vordefinierten Proteininteraktionsnetzwerk zu messen. Durch die gleichzeitige Durchführung mehrerer Co-IPs im selben Lysat können Bioinformatik-Techniken dann untersuchen, wie sich Gruppen von Co-Verbänden koordiniert verändern. Die Entwicklung des Assays ist zeitaufwändig, aber sobald das QMI-Panel in der Hand ist, können Netzwerk-Skala-Daten über Signaltransduktionssysteme in einem relativ einfachen Nachttest gesammelt werden.

QMI erfordert die genaue Pipettierung verschiedener Proben und Reagenzien in 96 Bohrplatten. Beim Einrichten und Ausführen jedes Assays sollten sorgfältige Hinweise gemacht werden, um Fehler zu vermeiden. Für die Probenvorbereitung und Immunpräzipitation beginnen Sie mit der Lysierung der Probe in einem geeigneten Reinigungsmittel mit Protease- und Phosphatase-Inhibitoren für eine 15-minütige Inkubation auf Eis.

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