Western blotting is a method of transferring proteins to a membrane and then using antibodies for detection. For the known expressed protein, the corresponding antibody can be used as the primary antibody to detect, and the expression product of the new gene can be detected by the antibody of the fusion part.
Similar to the Southern or Northern hybridization method, Western Blot uses polyacrylamide gel electrophoresis, and the object to be detected is a protein, the probe is an antibody, and the secondary antibody used for color development is labeled.
Western blotting, also known as immunoblotting, is an immunochemical technique that can detect proteins immobilized on a solid support. The protein to be tested can be either crude extract or a certain separation and purification. In addition, the application of this technology requires the use of monoclonal or polyclonal antibodies to identify the protein to be tested.
The soluble antigen, that is, the protein to be tested must first be separated by different electrophoresis methods according to its properties, such as molecular weight, molecular size, charge, and its isoelectric point; the protein in the gel is transferred to the polyvinylidene fluoride membrane; using the principle that the antibody (primary antibody) specifically binds to the antigen, the antibody is used as a probe to catch the target protein. It is worth noting that non-specific protein should be added before adding the primary antibody, such as bovine serum albumin to "block" the membrane to prevent non-specific binding of the antibody to the membrane.
Proteins separated by electrophoresis often need to use electrophoresis to transfer the protein to the solid phase carrier. We call this process electrophoretic blotting. The two commonly used electrical transfer methods are:
Semi-dry method: the gel and the solid phase carrier are sandwiched between filter papers soaked with a buffer solution, and the power-on time is 10 to 30 minutes.
Wet method: The gel and solid carrier are immersed in the transfer buffer solution, and the transfer time can be extended from 45 minutes to overnight.
Because the use of the wet method is more flexible and does not significantly waste more time and raw materials, we only describe the basic operation process of the wet method here.
The identification of the target protein requires the use of a secondary antibody that recognizes the primary antibody. The antibody is often a purchased product that has been bound or labeled with a specific reagent, such as horseradish peroxidase. This label is a colorimetric reaction catalyzed by horseradish peroxidase. The product of the reaction has a specific color and is fixed on a solid support for easy identification. Therefore, the primary antibody can be identified by identifying the secondary antibody, and then the location of the target protein can be determined. Other recognition systems include alkaline phosphatase system and 125I labeling system.
Protein sample preparation
1). Extraction of total protein from monolayer adherent cells
2). Extraction of total protein from tissue
3). Extraction of total protein from adherent cells treated with drugs
Determination of protein content
1). Make a standard curve
2). Test the protein content of the sample
1). Clean the glass plate
2). Pouring and loading
Chemiluminescence, imaging, fixing
Gel image analysis
It has the advantages of high resolution of gel electrophoresis, high specificity of solid phase immunity and high sensitivity, and can be used for qualitative, quantitative analysis and molecular weight determination of protein/peptide antigens.
It can detect monoclonal antibodies from polyclonal antibodies and determine the specificity of polyclonal antibodies; detect specific antigens from promiscuous antigens and provide information that ELISA cannot provide.
In the field of allergies, immunoblotting is suitable for studying the characteristics, standardization and cross-allergenicity of allergens.
The immunoblotting method is suitable for various laboratories because of its convenient and simple operation, and can be widely used in basic and clinical trials.
The wet immobilized matrix membrane is flexible and easy to handle;
The immobilized biocopy macromolecules can be uniformly close to various immunoprobes, and will not be blocked by pores like gels;
Only a few reagents are needed for immunoblotting analysis;
The time of incubation and washing is significantly reduced;
Multiple copies can be made at the same time for various analysis and identification;
The results can be stored for a long time in the form of graphs;
Immunoprobes can be used to reduce the pH value and other methods, like erasing the recording tape, and then wipe off the probe, and then use a second probe for analysis and detection. The application scope and advantages of immunoblotting are not limited to this, it will surely continue to develop and improve with the in-depth study of this method.