The Biology Department of Shanghai Medicilon has extensive experience in the field of in vitro biology, through enzyme level determination, cell level determination, cell biology, biochemistry, in vitro isotope determination, stable cell line establishment, gene knockout, RNAi and MicroRNA Technology, etc., to provide a complete set of biological services.
In cell biology research, sometimes it is necessary to qualitatively or/and quantify cell membrane molecules, intracellular molecules or expression products. However, since the expression level of the target protein is not too high, it is difficult to achieve the experimental purpose by SDS-PAGE electrophoresis and Western blot detection with cell lysate or expression supernatant. For this purpose, antibodies that specifically recognize the target protein can be used for immunoprecipitation, purification and enrichment of the target antigen. The protein obtained by immunoprecipitation can be subjected to SDS-PAGE electrophoresis, and the target protein bands can be observed after Coomassie brilliant blue staining or silver staining. If the amount of target protein obtained by immunoprecipitation is lower than the detection limit of Coomassie brilliant blue or silver staining, Western blot detection method can be used to improve the sensitivity of detection and achieve the experimental purpose.
Immunoblotting, also known as western blot, is a method for detecting a certain protein in a complex sample based on the specific binding of antigen and antibody. Western blot is a new immunobiochemical technology developed on the basis of gel electrophoresis and solid-phase immunoassay technology. Because of the high resolution of SDS-PAGE and the high specificity and sensitivity of solid-phase immunoassay, western blotting has become a routine technique for protein analysis. Western blotting is often used to identify a certain protein, and can perform qualitative and semi-quantitative analysis of the protein.
The immunoblotting method is performed in three stages.
The first stage is SDS-polyacrylamide gel electrophoresis (SDS-PAGE):
protein samples such as antigens are negatively charged after SDS treatment, and migrate from the cathode to the anode in the polyacrylamide gel. The smaller the molecular weight, the migration The faster the speed. The separation effect at this stage is not visible to the naked eye (the electrophoretic zone is only visible after staining).
The second stage is electrotransfer:
transfer the separated bands in the gel to the nitrocellulose membrane, select low voltage (100V) and high current (1~2A), and the transfer can be completed after powering on for 45 minutes. The protein bands separated at this stage are still invisible to the naked eye.
The third stage is enzyme immunolocalization:
the nitrocellulose membrane (corresponding to the solid phase carrier coated with the antigen) printed with protein bands is sequentially reacted with specific antibodies and enzyme-labeled secondary antibodies, and then added to form an insoluble display. The enzyme reaction substrate of the color substance, which makes the zone dyed.
The principle of protein immunoassay
Blotting method: transfer the biological macromolecules to the solid-phase carrier through different ways. The transferred solid-phase carrier is treated with quenching reagent, rinsed with an appropriate solution, and incubated in the solution containing the substrate or probe , Can react with the corresponding probe, showing a specific band.
Common imprinting methods are Southern, Northern, and Western blotting methods, which are used to detect DNA, RNA, and protein, respectively. Among them, Western blotting is also called Western blotting.
Among them, the basic principle of immunoblotting is to combine gel electrophoresis with solid-phase immunity, and its main operations are divided into three processes:
1) Electrophoresis: SDS-PAGE separates the protein components;
2) Imprinting: transfer the protein from the gel to the solid phase carrier;
3) Immunoassay: It reacts with the primary antibody and the labeled secondary antibody in sequence, and the color is excited by the substrate or excitation light, and the presence or absence of the target band is observed.
Western Blotting is a technique used to detect proteins.
First, the protein mixture containing the protein to be tested is separated by gel electrophoresis, and then the separated protein is transferred from the gel to a solid support by electrophoresis technology. This solid support is currently usually a nitrocellulose membrane. Subsequently, an antibody specific to the antigenic determinant of the protein to be tested (called the first antibody) is used as a probe to perform an immunological reaction with the protein on the solid support, and finally with horseradish peroxidase or alkaline phosphate. The esterase anti-primary antibody (second antibody) immunoreacts with the primary antibody, and only the test protein that specifically binds to the primary antibody can react with the secondary antibody. A color reaction occurs when the substrate with horseradish peroxidase is used as a color developing agent, and the test protein bound with the first antibody and the second antibody can be revealed through the color reaction.
The electrophoresis transfer of proteins from the gel to the solid support is accomplished by an electrotransfer instrument. It is to combine the solid support facing the anode and the gel facing the cathode, and then combine the two outer sides with Whatman 3MM filter paper to form a “sandwich” structure, put it on the electrotransfer instrument, and add electrophoresis buffer After the power is turned on, the protein can be transferred from the gel to the solid support.
Protein immune procedure
Put on gloves, remove the SDS-PAGE gel, and carefully peel off the gel. Cut out a nitrocellulose film the same size as the gel and six layers of Whatman 3MM filter paper, and cut off a corner at the lower left corner of the nitrocellulose film as a mark. Float the nitrocellulose membrane on the surface of deionized water, soak the water from the lower part of the membrane to remove the bubbles in it, soak the Whatman 3MM filter paper in the electro-transfer buffer, rinse the graphite electrode with distilled water, and soak the three layers first Place the wet filter paper on the anode, roll the glass rod on the surface of the filter paper to remove the air bubbles in between, and then carefully spread the soaked nitrocellulose film on the filter paper, and carefully remove the air bubbles with the glass rod. Then spread the 12% SDS-PAGE gel flat on the nitrocellulose film, carefully remove the bubbles with a glass rod, and then spread the three layers of soaked filter paper flat on the gel to remove the bubbles, and finally add graphite The electrode plate is connected to the power supply for electrical transfer, and the current is determined by the size of the gel, which is 0.65mA/cm². After 2 hours of electrotransfer, stop energizing, remove the electrotransfer device, and remove the gel for Coomassie brilliant blue staining to check whether the electrotransfer is complete. Carefully remove the nitrocellulose film and place it on a piece of dry filter paper. After 30-60 minutes of blotting dry, the color reaction begins.
First, the molecular weight marker is stained with amino black. Cut out the nitrocellulose film with molecular weight markers, rinse it with PBS buffer containing 0.3% Tween 20 for three times (15 minutes each time), then immerse it in the amino black dye solution, dye it at room temperature for 5 minutes, and then place it. Remove the background in amino black decolorizing solution, rinse in water and dry the scene for later use.
The nitrocellulose film transferred with the sample was blocked with PBS buffer containing 5% skim milk at room temperature for 2 hours, and rabbit anti-GST primary antibody was added 1:100 at the same time, shaking gently, and incubating at room temperature for 1 hour. Then rinse the nitrocellulose film 4 times with PBS buffer for 30 minutes each time. After the horseradish peroxidase-labeled goat anti-rabbit immunoglobulin secondary antibody was diluted 50 times with blocking solution, Tween 20 was added to a final concentration of 0.05%, and then reacted with the nitrocellulose film rinsed above. Dip the membrane in it, place it flat on a shaking table platform, incubate at room temperature for 1 hour, rinse the nitrocellulose membrane 4 times with PBS buffer for 5 minutes each time, and then add 4-chloronaphthol to display it. Color solution, add 5μ1 of 30% H2O2, place the nitrocellulose film in it and shake it slowly. After the detected protein bands reach the deepest degree of color development, transfer the nitrocellulose film to the PBS solution to stop the color reaction. Take out the nitrocellulose film, dry it naturally and take a picture to save the result.
Precautions for Western Blotting Technology
- The immunoblotting technique only measures the relative content of the target protein. Therefore, it is not possible to determine how much target protein a cell contains, only the presence or absence of the protein and whether the protein content is high or low compared with other cells under other conditions.
- When comparing the relative content of a target protein in different cells or the same cell under different conditions, the total protein content of each sample must be equal.
- Choose an appropriate concentration of SDS-PAGE gel so that the target protein can be better resolved.
- To start electrophoresis and transfer, be sure to confirm whether the positive and negative electrode connections are correct.
- In immunoassays, pay attention to blocking non-specific binding sites.
- The antibody reaction is carried out in a sealed plastic bag. All cheongsam in the bag must be taken out, otherwise it will cause uneven antibody binding.
- The operation should be gentle, wear gloves, and don’t transfer and apply any scratches. In the entire operation, the transfer film must always be in the liquid and not dry.