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Western blot analysis
Expression levels of Akt and phosphorylated Akt in tumor mice
Western blotting (Westernblotting) is a hybrid technique that combines high-resolution gel electrophoresis and immunochemical analysis techniques. Western blotting has the advantages of large analysis capacity, high sensitivity and strong specificity. It is one of the most commonly used methods for detecting protein characteristics, expression and distribution, such as qualitative and quantitative detection of tissue antigens, quality determination of peptide molecules and viral Antibody or antigen detection, etc. Immunoblotting is commonly used to identify a certain protein, and can perform qualitative and semi-quantitative analysis of the protein.
The methods of Western Blot color development are as follows:
Autoradiography
Substrate chemiluminescence ECL
Substrate fluorescence ECF
Substrate DAB is colored
Basic steps of immunoblotting:
SDS-PAGE is the most commonly used electrophoresis method for qualitative analysis of proteins, especially for protein purity detection and determination of protein molecular weight. PAGE can effectively separate proteins, mainly based on the difference in molecular weight and charge, while SDS-PAGE (SDS denaturation does not The separation principle of continuous polyacrylamide gel electrophoresis is only based on the difference in molecular weight of the protein, because the sample processing solution of SDS-PAGE is added to the sample to be run for electrophoresis containing SDS, which can break cysteine residues. The disulfide bond between them destroys the quaternary structure of the protein. SDS is an anionic surfactant or detergent, which can break the intramolecular and intermolecular hydrogen bonds and destroy the secondary and tertiary structures of protein molecules. , And combined with the hydrophobic part of the protein, destroying its folded structure. After adding the electrophoresis sample to the sample buffer, it should be boiled in boiling water for 5 minutes to denature so that SDS and protein are fully combined to form an SDS-protein complex. When the strong reducing agent mercaptoethanol is present, the disulfide bond in the protein molecule is opened without being oxidized, the protein is completely denatured and depolymerized, and a rod-like structure is formed, which is stable in a uniform solution. After the SDS is combined with the protein, the SDS -The protein complex carries a large amount of negative charge, and on average every two amino acid residues are combined with an SDS molecule. At this time, the charge of each protein molecule is completely covered by SDS, far exceeding its original charge, so that The original charge of the protein is negligible, eliminating the original charge difference between different molecules. Its electrophoretic mobility mainly depends on the size of the molecular weight of the subunit, so the separated band is also the subunit of the protein. Sample Bromophenol blue dye is usually added to the treatment solution. Bromophenol blue indicator is a small molecule that can freely pass through the pore size of the gel, so it shows the leading position of electrophoresis. When the indicator reaches the bottom of the gel, it can be stopped. Electrophoresis. In addition, an appropriate amount of glycerin or sucrose can be added to the sample processing solution to increase the density of the solution, so that the sample solution can sink to the bottom of the sample adding tank during sample addition. The most common gel electrophoresis buffer consists of Tris-glycine. The buffer may contain 0.1% detergent, usually SDS. Tris-glycine can be used to separate proteins in a wide molecular weight range (6-200 kDa) and is compatible with denatured or non-denatured conditions.
detection and determination of protein molecular weight. PAGE can effectively separate proteins, mainly based on the difference in molecular weight and charge, while SDS-PAGE (SDS denaturation does not The separation principle of continuous polyacrylamide gel electrophoresis is only based on the difference in molecular weight of the protein, because the sample processing solution of SDS-PAGE is added to the sample to be run for electrophoresis containing SDS, which can break cysteine residues. The disulfide bond between them destroys the quaternary structure of the protein. SDS is an anionic surfactant or detergent, which can break the intramolecular and intermolecular hydrogen bonds and destroy the secondary and tertiary structures of protein molecules. , And combined with the hydrophobic part of the protein, destroying its folded structure. After adding the electrophoresis sample to the sample buffer, it should be boiled in boiling water for 5 minutes to denature so that SDS and protein are fully combined to form an SDS-protein complex. When the strong reducing agent mercaptoethanol is present, the disulfide bond in the protein molecule is opened without being oxidized, the protein is completely denatured and depolymerized, and a rod-like structure is formed, which is stable in a uniform solution. After the SDS is combined with the protein, the SDS -The protein complex carries a large amount of negative charge, and on average every two amino acid residues are combined with an SDS molecule. At this time, the charge of each protein molecule is completely covered by SDS, far exceeding its original charge, so that The original charge of the protein is negligible, eliminating the original charge difference between different molecules. Its electrophoretic mobility mainly depends on the size of the molecular weight of the subunit, so the separated band is also the subunit of the protein. Sample Bromophenol blue dye is usually added to the treatment solution. Bromophenol blue indicator is a small molecule that can freely pass through the pore size of the gel, so it shows the leading position of electrophoresis. When the indicator reaches the bottom of the gel, it can be stopped. Electrophoresis. In addition, an appropriate amount of glycerin or sucrose can be added to the sample processing solution to increase the density of the solution, so that the sample solution can sink to the bottom of the sample adding tank during sample addition. The most common gel electrophoresis buffer consists of Tris-glycine. The buffer may contain 0.1% detergent, usually SDS. Tris-glycine can be used to separate proteins in a wide molecular weight range (6-200 kDa) and is compatible with denatured or non-denatured conditions.
Immunoblotting was performed in three stages: SDS-polyacrylamide gel electrophoresis (SDS-PAGE) -> electrotransfer -> enzyme immunolocalization. The specific operation process of immunoblotting detection is very different. The most common of these are direct and indirect detection. Indirect detection means that the primary antibody first binds to the antigen, and then adds secondary antibodies that can specifically bind to the primary antibody. Labels include biotin, fluorescent probes (such as rhodamine) and enzymes (such as HRP and AP). The advantage of this method is that a single secondary antibody can measure a variety of primary antibodies to avoid labeling the primary antibodies one by one. On the other hand, the primary antibody can usually bind to several secondary antibody molecules, which plays a role in signal amplification. . The disadvantage is that the detection process adds an additional incubation step.
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