Abstract: The biotin-avidin system (BAS) is a new type of biological reaction amplification system developed in the late 1970s. With the advent of various biotin derivatives, BAS has been widely used in various fields of medicine. A large number of studies in recent years have confirmed that the biotin-avidin system can be combined with almost all kinds of markers successfully studied. The strong combination of high affinity between biotin and avidin and multi-stage amplification effects makes BAS immunolabeling and related tracer analysis more sensitive. Biotin is widely distributed in animal and plant atissues and is often extracted from high content yolk and liver tissue. After chemical modification, biotin can be a derivative with a variety of reactive groups - activated biotin. Activated biotin can be conjugated to almost all known biomacromolecules, including proteins, nucleic acids, polysaccharides, lipids, etc., mediated by protein cross linkers. Generally, we call this activated biotin for marking a biotin marker. This technology has been widely used in the field of micro-antigen, antibody qualitative, quantitative detection and localization observation. Of course, there is also a certain application of granulocyte marker, but this article will not discuss in detail.

Keywords: biotin marker, gene probe, protein



u Biotin markers and gene probe
Traditional gene probes are synthesized by radioactive phosphate bases. We can synthesize gene probes with biotin-marked phosphate bases to avoid possible damage from radioactive materials during the experiment.





For example, it has been proved that the synthesized biotin-marked cDNA probe can be well used for the detection of a large amount of apple rust virus, and has broad application prospects. The detection of apple rust fruit virus by biotin-marked cDNA probe has three advantages:

Low cost, high sensitivity and specificity. RT-PCR technology is suitable for the detection of small samples in the laboratory, while biotin-marked cDNA probes can detect a large number of samples.
The markers do not involve the enzyme system and the complex reaction system. The prepared probe can be stored for a long time and is suitable for the preparation of a large number of probes in the standardized kit.
No radioactive contamination.


u Analysis of cell membrane proteins with biotin markers
About one-third of the genes encoded by the human genome are cell membrane proteins, which play important roles in many life activities, such as cell recognition, signal transduction, and material transport. Due to the accessibility of the plasma membrane, many membrane proteins have become important targets for drug therapy. However, the difficulty from the separation and purification of the plasma membrane fraction, the low abundance of membrane protein, and the strong hydrophobicity pose challenges to isolation, identification and functional analysis. As the main technology for large-scale separation of complex proteins, traditional two-dimensional gel electrophoresis cannot be well applied to the separation and identification of cell membrane proteins.

In order to improve purification efficiency and minimize contamination, biotin marking can be used to selectively separate and purify cell membrane proteins, which is the basis for quantitative analysis of membrane proteins by amino acid stable isotope marking in cell culture.

The main steps are as follows: 1. In situ labeling of intact cell surface proteins using the membrane-impermeable biotin reagent sulfo-NHS-IC-biotin. 2. Cell lysis assay. 3. Streptavidin affinity purification of biotinylated protein, trypsin digestion. 4. Finally, the protein is identified by LC-MS/MS. This method of selectively enriching membrane proteins by biotinylation and subsequent affinity purification techniques has been used for high-throughput extraction of membrane proteins, but membrane proteins obtained after affinity purification still contaminate some cytoplasmic proteins.

Biotin interacts with avidin to affinity-purify biotinylated proteins and digests these proteins, which increases purification efficiency and reduces contamination. Cell surface biotinylation has become an important tool for studying various membrane proteins, such as the expression and regulation of receptors and transporters, and the distribution of membrane proteins in polar epidermal cells.

u Role in the process of protein purification
What is affinity chromatography?
Affinity chromatography is to place an affinity molecule with a special structure as a solid phase adsorbent in a chromatography column. When the protein mixture to be separated passes through the column, the protein having affinity with the adsorbent is adsorbed and retained in the column. Those proteins without affinity are directly eluted because they are not adsorbed, and are separated from the separated proteins, and then the appropriate eluent is used to change the binding conditions to elute the bound proteins.

How to improve the purity process of protein?
The biotin-avidin system can be combined with affinity chromatography to greatly increase the purity of the purified protein or to find a receptor for a known ligand. The step is to first covalently bind biotin to the ligand protein, then add the biotinylated ligand protein to the mixture containing the receptor protein, and then pass the mixture through a column in which avidin is immobilized in advance. The ligand-receptor complex resides on the column via the biotin-avidin system, and finally the receptor ligand protein complex or only the receptor is obtained by selective elution. This method is widely used in the drug development industry. When a drug molecule is found to be therapeutic but it is unclear which protein it acts on, it can be biotinylated and then separated from thousands of proteins.

u Advantages of the BAS system


Biotin easily binds to biological macromolecules such as proteins and nucleic acids, and the biotin derivative forms not only maintain the original biological activity of the macromolecular substance, but also have higher specificity and more valence.
The affinity constant of avidin binding biotin can be a million times of the antigen-antibody reaction, and the dissociation constant of the combination of the two forms is small, irreversible reactivity; simultaneously acid, alkali, denaturing agent, protein Both lytic enzymes and organic solvents do not affect their binding. Therefore, in practical applications, the high stability can help reduce the operation error and improve the accuracy of the measurement.
The versatility of the biotin-avidin system also provides a uniform methodology. For example, for a molecule to be tested, a biotin-marked antigen for the molecule has been obtained, and the colloidal gold in combination with avidin can be observed under an electron microscope, and the fluorescently marked avidin can be selected by flow cytometry. An immunohistochemistry experiment such as ELISA can be performed with the avidin linked to the enzyme.
References   
[1] ZHAO Ying and NIU Jian-xin, Apple Scar Skin Viroids Detection by cDNA Probe Marked with Biotin. 1004-1389(2008)03-0302-06

[2] LU Jing, SHI Qian. Analysis of plasma membrane proteins of MCF-10A by cell surface biotinylation. Fudan Univ J Med Sci, 2010 Nov, 37(6)