Common Methods for Assessing Protein-Protein Interactions

Proteins are important biological molecules in the body, and their functions are mainly realized through interactions. Interactions between proteins play an important role in biological processes such as cell signal transmission, metabolic regulation, immune response, etc. Understanding the interactions between proteins is of great significance for biological pharmaceutical research and drug development.

 

Methods to Judge Protein Interactions

1. Co-Immunoprecipitation (Co-IP)

Co-Immunoprecipitation is a commonly used method to judge protein interactions. This method uses a specific antibody to bind with the target protein, then precipitates the target protein and its interacting protein complex using fixed protein A/G magnetic beads or agarose particles. Finally, the protein composition in the complex is detected by techniques such as Western blotting, thereby revealing the interaction relationship between the proteins.

 

2. Yeast Two-Hybrid (Y2H)

The yeast two-hybrid is a high-throughput protein interaction screening technology. This method uses the transcription activation domain and DNA binding domain in yeast cells to identify protein interactions. When two proteins interact, the transcription activation domain and the DNA binding domain combine, thereby triggering the expression of the reporter gene. This method can efficiently screen a large number of protein interaction partners.

 

3. Protein Complementation Assay (PCA)

The Protein Complementation Assay is a method that uses divided protein tags to recombine into active tags in cells. By fusing the two complementary tags of the target protein on two different vectors, and then co-transfecting them into cells, if the target proteins interact, the two tags will recombine to form a complete active tag.

 

4. Protein Cross-Linking

Protein cross-linking is a chemical cross-linking technique that can cross-link interacting proteins together through a cross-linking agent. The cross-linked protein complex can be detected by methods such as SDS-PAGE or mass spectrometry to determine the interacting proteins.

 

5. Fluorescence Resonance Energy Transfer (FRET)

FRET is a microscopic technique for real-time monitoring of protein interaction. This method uses fluorescently labeled protein pairs to study the distance and interaction between proteins. When two fluorescently labeled proteins are close, energy transfer causes changes in the fluorescence signal, thereby revealing the interaction state of the proteins.

 

Choosing Suitable Methods

1. Protein Characteristics and Purpose

Choosing a suitable method to judge protein interaction needs to consider the characteristics of the protein and the purpose of the experiment. For example, if the goal is to screen partners that interact with a specific protein, high-throughput techniques such as yeast two-hybrid are more suitable choices. If you need to observe protein interactions in cells, Protein Complementation Assay and FRET techniques are more applicable.

 

2. Experimental Conditions and Cost Considerations

Experimental conditions and cost are also factors to consider when choosing methods. Some techniques require complex experimental conditions and equipment and are costly, while others are simpler and more economical.

 

The interaction between proteins is a complex regulation network in the body and there are various methods to judge it. The suitable judgment method needs to consider factors such as protein characteristics, experimental purposes, and experimental conditions. By reasonably choosing applicable technical means, we can deeply reveal the mystery of protein interactions and provide an important reference for biological pharmaceutical research and drug development.