Protein Interaction Experiment Analysis: From Sample Preparation to Interaction Identification

Proteins are vital functional molecules in living organisms, participating in numerous biological processes through their interactions. Understanding these interactions between proteins is crucial in revealing mechanisms of signal transmission and metabolic regulation within organisms. Protein interaction experiments are one of the key methods to study these interactions. This article will discuss the entire process, from sample processing to interaction identification. It aims to help readers understand the basic principles and common methods of protein interaction experiments.

 

Sample Processing

Before carrying out protein interaction experiments, it is necessary to process the samples. The purpose of sample processing is to extract proteins while maintaining their original structure and function. Common sample processing methods include cell lysis, tissue sectioning, and serum separation. Cell lysis is the most common sample processing method, through which cells are disrupted and proteins are released by mechanical crushing, ultrasonic treatment, or chemical lysis. Tissue sectioning involves cutting tissue samples into thin sections and then extracting proteins. Serum separation involves separating proteins from the blood through centrifugation.

 

Detection Methods for Protein Interactions

There are various detection methods for protein interactions, including immunoprecipitation, yeast two-hybrid, surface plasmon resonance, and mass spectrometry. Each of these methods has its advantages and disadvantages, and the appropriate method can be chosen based on the experimental requirements.

 

1. Immunoprecipitation

Immunoprecipitation is a common method for detecting protein interactions. It involves binding a specific antibody to the target protein, and then co-precipitating proteins interacting with the target protein. By examining the proteins in the precipitate, the interacting partners of the target protein can be identified.

 

2. Yeast Two-Hybrid

The yeast two-hybrid is a protein interaction detection method based on yeast cells. This method uses transcription factors in yeast cells divided into two parts, one binds to the target protein, and the other to the promoter. When the target protein interacts with its partner, the two parts of the transcription factor recombine, thereby activating the expression of the reporter gene.

 

3. Surface Plasmon Resonance

Surface plasmon resonance is a protein interaction detection method based on optical principles. This method involves fixing the target protein on a metal film and using light reflection and transmission to detect protein binding. By measuring changes in reflected and transmitted light, the interaction strength and affinity of proteins can be determined.

 

4. Mass Spectrometry

Mass spectrometry is a high-sensitivity method for detecting protein interactions. This method involves separating and ionizing the proteins in the sample, and then detecting and analyzing the ions with a mass spectrometer. By analyzing the mass spectra, the interacting partners and binding sites of proteins can be identified.

 

Protein interaction experiments are an important means to study protein interactions, which can help us understand mechanisms of signal transmission and metabolic regulation in organisms. When conducting protein interaction experiments, it is necessary to process samples and choose suitable detection methods. Common detection methods include immunoprecipitation, yeast two-hybrid, surface plasmon resonance, and mass spectrometry.