Study on the technique of protein interaction analysis
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To explore the molecular mechanisms of biological processes, it is necessary to identify protein-protein interactions that mediate this process. The main techniques for studying protein-protein interactions are summarized as follows:
First, the yeast two-hybrid system
The yeast two-hybrid system is an important method currently widely used in the study of protein interaction omics. The principle is that when the target protein and the bait protein specifically bind, the bait protein binds to the promoter of the reporter gene, and initiates the expression of the reporter gene in the yeast cell. If the expression product of the reporter gene is detected, it means that there is mutual mutual Role, otherwise there is no interaction between the two.
This technique can be used for the study of large-scale protein interactions after being micro-sized and arrayed. In practice, one-dimension systems, three-hybrid systems, and reverse hybrid systems have been developed as needed. Angermayr et al. designed a SOS protein-mediated two-hybrid system. The function of membrane proteins can be studied to enrich the function of the yeast two-hybrid system. In addition, the role of the yeast two-hybrid system has also expanded to the identification of proteins.
Second, phage display technology
A DNA sequence encoding a monoclonal antibody encoding a phage coat protein gene, when the phage grows, the surface expresses the corresponding monoclonal antibody, and then the phage is passed through the column, and if the target protein is contained on the column, the antibody is specific to the corresponding antibody. In combination, this is called phage display technology.
This technology is also mainly used to study the interaction between proteins, not only has high-throughput and simple features, but also has direct access to genes, highly selective screening of complex mixtures, and can directly evaluate each other by appropriately changing conditions during the screening process. The advantages of binding specificity.
Currently, cDNA libraries of two specific cell lines of human and murine have been demonstrated using optimized phage display technology, and signal molecules in the human epidermal growth factor signaling pathway have been isolated.
Third, plasma resonance technology
Surface Plasmon Resonance (SPR) has become a new tool in the study of protein interactions. Its principle is to use a nano-scale film to adsorb the "bait protein". When the protein to be tested is combined with the bait protein, the resonance property of the film changes, and the combination of the two proteins can be known through detection. The advantage of SPR technology is that no label or dye is required and the reaction process can be monitored in real time. The assay is fast and safe and can also be used to detect interactions between protein-nucleic acids and other biomacromolecules.
Fourth, fluorescent energy transfer technology
Fluorescence resonance energy transfer (FRET) is widely used to study the distance between molecules and their interactions; combined with fluorescence microscopy, quantitative information on the temporal and spatial information of proteins, lipids, DNA and RNA in living organisms.
With the development of green fluorescent protein (GFP), it is possible for FRET fluorescence microscopy to measure the dynamic properties of molecules in living cells in real time. A simple method for quantitatively measuring the FRET efficiency and the distance between the donor and the acceptor is proposed by using a set of filters and measuring a ratio, using the emission spectra of the donor and acceptor to eliminate crosstalk between the spectra. The method is simple and rapid, and can quantitatively measure the efficiency of FRET and the distance between donor and acceptor in real time, and is especially suitable for GFP-based donor receptor pairs.
V. Antibody and protein array technology
The emergence of protein chip technology brings new ideas to proteomics research. A major part of proteomics research is to study the quantitative change, miniaturization and integration of protein levels under different physiological conditions. Qualcomm quantified antibody chips are a very good research tool, and he is also the fastest growing chip in the chip. And it has become increasingly mature in technology. Some of these antibody chips have been developed for clinical applications, such as tumor marker antibody chips, and many other fields that have been applied again.
Sixth, immunoprecipitation technology
Co-immunoprecipitation is mainly used to study the interaction between protein and protein. The basic principle is to add anti-protein antibody to the cell lysate, and then add the specific binding to the antibody to the Pansobin beads after incubation. Staphylococcus aureus protein A (SPA), if the cell has a protein of interest that is bound to the protein of interest, a complex can be formed: "protein of interest - protein of interest - anti-interest protein antibody - SPA \ | Pansobin", Because SPA\|Pansobin is relatively large, the complex is separated during centrifugation.
After denaturing polyacrylamide gel electrophoresis, the four components of the complex were separated. Then, by Western blotting, the antibody is used to detect what the protein of interest is and whether it is a predicted protein. The target protein obtained by this method is naturally bound to the protein of interest in the cell, and conforms to the actual situation in the body, and the obtained protein has high credibility.
However, this method has two drawbacks: First, the combination of the two proteins may not be directly combined, and there may be a third party acting as a bridge in the middle; second, it is necessary to predict what the target protein is before the experiment to select the final detection. Antibodies, so if the prediction is not correct, the experiment will not get results, and the method itself is risky.
Seven, pull-down technology
There are two types of protein interactions: strong interactions and transient interactions. Strong interactions are common with multi-subunit protein complexes, preferably by co-immunoprecipitation (Co-IP), Pull-down techniques, or Far-western methods. The Pull-down technique uses a immobilized, labeled bait protein or tagged protein (Biotin-, PolyHis- or GST-) to capture proteins that interact with it from cell lysates.
The Pull-down technique can be used to determine the interaction between a known protein and a cloned protein or a purified related protein, and to detect protein interactions from an in vitro pathway or translation system.
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