Visualization of Protein-protein Interaction in Nuclear and Cytoplasmic Fractions by Co-immunoprecipitation and <em>In Situ</em> Proximity Ligation Assay

Xinzhou Zhu; Andrea Zelmer; Sven Wellmann

doi:10.3791/55218

Visualisation des interaction protéine-protéine en nucléaire et cytoplasmique Fractions par Co-im...

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Biochemistry

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JoVE Journal Biochemistry

Visualization of Protein-protein Interaction in Nuclear and Cytoplasmic Fractions by Co-immunoprecipitation and In Situ Proximity Ligation Assay

Visualisation des interaction protéine-protéine en nucléaire et cytoplasmique Fractions par Co-immunoprécipitation et In Situ Proximité Ligation Assay

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10:05 min

January 16, 2017

DOI: 10.3791/55218-v

Xinzhou Zhu¹, Andrea Zelmer¹, Sven Wellmann^1,2

¹University Children's Hospital Basel (UKBB),University of Basel, ²Department of Clinical Research,University of Basel

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Overview

This study investigates protein-protein interactions in the nucleus and cytoplasm using co-immunoprecipitation and proximity ligation assay. By comparing these methods, researchers can determine the most suitable approach for visualizing NF90-RBM3 interactions.

Key Study Components

Area of Science

Neuroscience
Cell Biology
Biochemistry

Background

Protein-protein interactions are crucial for cellular functions.
Understanding these interactions can provide insights into cellular mechanisms.
Different methods exist for studying these interactions, each with its advantages.
Co-immunoprecipitation and proximity ligation assay are two prominent techniques.

Purpose of Study

To compare co-immunoprecipitation and proximity ligation assay.
To evaluate their effectiveness in visualizing protein interactions.
To assist researchers in selecting the appropriate method for their studies.

Methods Used

Co-immunoprecipitation (co-ip) for detecting authentic interactions.
Proximity ligation assay (pla) for rapid analysis at the single-cell level.
Hex293 cell culture and extraction protocols.
Comparison of distribution patterns in cellular compartments.

Main Results

Co-ip effectively detects authentic protein interactions.
Pla allows for quick analysis of interactions at the single-cell level.
Both methods provide valuable insights into protein distribution.
The choice of method depends on the specific research goals.

Conclusions

Both co-ip and pla have unique advantages for studying protein interactions.
Researchers should consider their specific needs when choosing a method.
This study aids in understanding the distribution of NF90-RBM3 interactions.

Frequently Asked Questions

What are the main advantages of co-immunoprecipitation?

Co-immunoprecipitation is effective for detecting authentic protein-protein interactions.

How does proximity ligation assay differ from co-immunoprecipitation?

Proximity ligation assay allows for rapid analysis at the single-cell level, unlike co-immunoprecipitation.

What cell type was used in this study?

Hex293 cells were used for the experiments.

Why is it important to study protein-protein interactions?

Studying these interactions helps to understand cellular mechanisms and functions.

Can both methods be used simultaneously?

While they can be used in conjunction, each method serves different purposes and may yield different insights.

What should researchers consider when choosing a method?

Researchers should consider the specific goals of their study and the advantages of each method.

Les interactions protéine-protéine peuvent se produire à la fois dans le noyau et le cytoplasme d’une cellule. Pour étudier ces interactions, la co-immunoprécipitation traditionnelle et le test moderne de ligature de proximité sont appliqués. Dans cette étude, nous comparons ces deux méthodes pour visualiser la distribution des interactions NF90-RBM3 dans le noyau et le cytoplasme.

L’objectif général de cette expérience est de comparer les avantages et les inconvénients de la co-immunoprécipitation, ou co-ip, et du test de ligature de proximité, ou pla, dans la visualisation des interactions protéiques dans différents compartiments cellulaires. La comparaison présentée ici de deux méthodes différentes peut aider les scientifiques à décider laquelle est la mieux adaptée à leur objectif spécifique lorsqu’ils étudient les modèles de distribution des interactions protéine-protéine dans le noyau et le cytoplasme d’une cellule. Le principal avantage de la co-IP est de détecter les interactions protéiques authentiques.

En revanche, l’avantage du pla est d’analyser rapidement les interactions protéiques de l’institut au niveau de la cellule unique. Après avoir cultivé et récolté des cellules hex293 selon le protocole de texte, à 3 fois 10 à la 6ème cellule, ajoutez 300 microlitres de solution d’extraction cytoplasmique froide et vortex les cellules à la vitesse la plus élevée pendant 15 secondes. Incuber l’extrait sur de la glace pendant 30 minutes, et toutes les 10 minutes pendant l’incubation, agiter l’échantillon pendant cinq secondes.

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