Molecular Biological Detection

1. Molecular Biological Detection With the continuous development of life science and chemistry, people's understanding of organisms has been gradually deepened to the micro level. From individual organisms to organs, tissues and cells, and from cellular structure to the molecular level of nucleic acids and protein structure, people realized that the linear structure at the molecular level could be examined to compare horizontally the differences between different species, different individuals from the same species, different cells from the same individuals, or different physiological (pathological) states. This provides a powerful technical platform for all fields of biology and medicine. Molecular biological detection technology is the crystal of great progress in modern molecular biology and molecular genetics. It is based on the humans’ deepening understanding of the structure of genes, gene expression and regulation and other essential issues of life. In recent years, great progress has been made in the methodological research of molecular biological detection technology, and various molecular biological detection technologies have been established successively. Here to introduce some methods in molecular biological detection: Co Immunoprecipitation Immunoprecipitation is a classical method for studying Protein-Protein Interaction based on the specific interaction between antibodies and antigens. It is an effective method to determine the endogenous interactions of two proteins in intact cells. The theory is to take the cell endogenous target protein as bait. Then The target protein antibody was co-incubated with the total cell protein to promote the formation of immune complexes. Subsequently, protein A/G that can bind to the target protein antibody was added to form the "binding protein-target protein-target antibody" complex. After purification, the immune complex was isolated by gel electrophoresis and combined with Western blot or mass spectrometry to achieve the purpose of protein purification and qualitative detection of antigen or antibody. Compared with other research methods, immunoprecipitation detects the interaction between proteins under physiological conditions. Therefore, it can not only detect the natural complex formed in the body, but also exclude false positives caused by overexpression of target proteins. Second, endogenous target proteins are fully processed, modified, and mature proteins, so modified dependent protein interactions can also be detected. Pull Down Assay This assay is an effective in vitro test technology to verify the yeast two-hybrid system. It is more and more popular among scholars in recent years. The basic principle is to bind the target protein -GST fusion protein to glutathione affinity resin, as a support for the target protein affinity, as a "decoy protein". Purpose protein solution get through column, then can capture the “capture protein" (purpose protein) which interact with protein solution , elution by sds-page electrophoresis analysis after the combination, which confirmed the interaction between two proteins or screening of the purpose of the corresponding protein. Both "bait

2. protein" and "capture protein" can be obtained by cell lysates, purified proteins, expression systems and in vitro transcription translation systems, etc. This method is simple and easy to operate. Far-Western Blot Far-western blot is a molecular biological method based on western blot, it is used to stain the cells or biological tissue samples treated by gel electrophoresis through specific antibodies, and to obtain information about the expression of specific proteins in the cells or tissues analyzed by analyzing the position and depth of coloring. Antibodies were used to detect the target protein in western blot, while non-antibody proteins that bind to the target protein were used in far-western blot. Therefore, western blotting is used to detect specific proteins while far-western blotting is used to detect interactions between proteins. Proteome research can not only provide a material basis for the regulation of life activities, but also provide theoretical basis and solutions for the elucidation and attack of various diseases. By comparing the proteome between normal individuals and pathological individuals, we can find some "disease-specific protein molecules" that can serve as molecular targets for the design of new drugs or provide molecular markers for the early diagnosis of diseases. Therefore, proteomics research is not only the necessary of explore the mysteries of life, but also can bring great benefits to human health. The study of proteomics is a feature of life science entering the post-gene era.