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Things you need to know about bovine serum albumin.
2019-03-25
来源：亚科官网
Bovine serum albumin is one of the most commonly used proteins in biochemical laboratories, and in experiments it may be overlooked because it is too common and too mundane. Bovine serum albumin (BSA), also known as the fifth component, is a globulin in bovine serum containing 583 amino acid residues with a molecular weight of 66.430 kDa and an isoelectric point of 4.7. Bovine serum albumin is widely used in biochemical experiments, for example as a blocking agent in western blot.
Structural characteristics of bovine serum albumin
The bovine serum albumin precursor protein is 607 amino acids in length. The precursor protein removes 18 signal peptides from the N-terminus and 6 propeptides to form a mature BSA protein of 583 amino acids with a molecular weight of approximately 66.5 kDa. The surface of BSA contains a large amount of acidic and basic amino acids. Among them, there are 40 and 59 acidic amino acids glutamic acid and aspartic acid, respectively, and 59 and 23 basic amino acids lysine and arginine. In addition, BSA has 35 cysteines, forming 17 pairs of disulfide bonds and a free sulfhydryl group.
Application of bovine serum albumin
1. Maintain the stability of protein
When the protein is under "pressure" conditions, such as heating, ultraviolet irradiation, oxidation, shearing force, etc., the spatial folding of the protein tends to spread, resulting in the exposure of internal hydrophobic sites, amorphous aggregation or formation of highly structured amyloid. Protein, which loses its biological activity. BSA has good thermal stability, and its spatial structure changes are still reversible when the temperature rises to 65 degrees. Another feature of BSA is its ability to form reversible bonds with many substances, especially poorly water-soluble substances such as fatty acids, heme, bilirubin, negatively charged aromatic compounds, etc., which increase its water solubility. Therefore, BSA is an ideal protein protectant. When a protein such as an enzyme or an antibody undergoes a spatial structural change, BSA can reversibly bind to it to form a stable, soluble polymer complex, preventing amorphous aggregation of the protein. In addition, the BSA in the solution can bind to the surface of the container, preventing the protein from being adsorbed on the surface of the container and decreasing in concentration.
As Chang et al. 1995 found, the stability of proteins is determined by the hydrophobicity of the protein surface. When the temperature rises, the hydrophobic site of the enzyme is exposed, and the BSA in the solution binds to the hydrophobic region of the enzyme, thereby maintaining the water solubility of the enzyme, preventing aggregation, and effectively improving the thermal stability of the enzyme. Moreover, the more hydrophobic the protective protein, the stronger the protective effect on the enzyme.
2. Solid phase blocking protein in immunoassay
During the immunoassay, the antibody needs to be immobilized on a solid surface such as a microplate, magnetic particles, polystyrene microspheres or a nitrocellulose membrane surface. Since the protein is a viscous molecule, the heteroprotein in the sample will non-specifically bind to the hydrophobic site on the surface of the solid phase during the detection process, causing background interference. Therefore, it is necessary to block the excess sites on the surface of the solid phase with a blocking agent. Commonly used blocking agents, such as BSA, casein, gelatin, etc., can be adsorbed on the surface of the solid phase to prevent non-specificity of the antibody or antigen and the surface of the solid phase. Combine to reduce the detection background and improve sensitivity.
BSA is a globulin with a molecular weight of about 66.5 kDa, a large amount of amino and carboxyl residues on the surface, and a spatial structure of BSA that changes with temperature, pH, and salt concentration. As a common blocking protein, BSA is ideal for binding to hydrophilic surfaces, hydrophobic surfaces, and activated covalently bonded surfaces, such as activated carboxyl groups, amino groups, epoxy groups, tosyl groups, etc., to inhibit non-specific adsorption.
The surface of BSA contains a large number of carboxyl groups and amino groups, which can be used for the binding of active groups on the surface to block excess sites. The most common application is the application of chemiluminescent immunomagnetic beads-conjugated antibodies. If the carboxyl surface magnetic beads are activated by EDC, they can react with the amino groups on the surface of the antibody to covalently bind the antibody to the surface of the magnetic beads. This process requires the blocking of excess activated carboxyl sites. The BSA molecule is smaller in size and can enter the gap between the surface antibodies, and because the surface contains a large amount of amino groups, it reacts with the activated carboxyl groups to block the excess active sites. At the same time, the surface of BSA contains a large amount of carboxyl groups, which can also improve the hydrophilicity of the magnetic beads. In addition, the surface of BSA contains certain amino acid hydrophobic residues, such as alanine, leucine, etc., and can also block the hydrophobic sites on the surface.
It is worth noting that Larsericsdotter et al. found that BSA is more compact in the phosphate solution on the solid surface and the structure is more stable. This is because phosphate binds to glycine and arginine on BSA, which eliminates structural electrostatic repulsion. Therefore, BSA is more stable in phosphate buffer and has higher binding efficiency on the solid surface.
Related links: Bovine serum albumin
Edited by Suzhou Yacoo Science Co., Ltd.
