Western Blot western blot experiment operation method
Western Blot (Western Blot) is to transfer the protein to the membrane, and then use antibodies to detect. 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.
1. Principle:
Similar to the Southern or Northern hybridization method, but Western Blot uses polyacrylamide gel electrophoresis, the object to be detected is a protein, the "probe" is an antibody, and the "color" is a labeled secondary antibody. The protein sample separated by PAGE is transferred to a solid-phase carrier (such as nitrocellulose membrane). The solid-phase carrier adsorbs proteins in the form of non-covalent bonds, and can maintain the type of polypeptide separated by electrophoresis and its biological activity. The protein or peptide on the solid-phase carrier is used as an antigen to react with the corresponding antibody, and then react with the enzyme or isotope-labeled secondary antibody. After the substrate color development or autoradiography to detect the specific purpose of electrophoretic separation The protein component of gene expression. This technique is also widely used to detect protein level expression.
Second, reagent preparation:
1. SDS-PAGE reagent: see electrophoresis experiment.
2. Homogenization buffer: 1.0M Tris-HCl (pH 6.8) 1.0ml; 10% SDS 6.0ml; β-mercaptoethanol 0.2ml; ddH2O 2.8ml.
3. Transfer membrane buffer: 2.9 g of glycine; 5.8 g of Tris; 0.37 g of SDS; 200 ml of methanol; add ddH2O to bring the volume to 1000 ml.
4. 0.01M PBS (pH7.4): NaCl 8.0g; KCl 0.2g; Na2HPO4 1.44g; KH2PO4 0.24g; add ddH2O to 1000ml.
5. Membrane staining solution: Coomassie brilliant blue 0.2g; methanol 80ml; acetic acid 2ml; ddH2O118ml. Coating solution (5% skimmed milk powder, now prepared): 1.0g of skimmed milk powder dissolved in 20ml of 0.01M PBS.
6. Color developing solution: DAB 6.0mg; 0.01M PBS 10.0ml; nickel amine sulfate 0.1ml; H2O2 1.0μl.
3. Operation steps:
1. Protein sample acquisition: After the bacteria induce expression, the cells can be directly lysed by electrophoresis loading buffer, eukaryotic cells plus homogenization buffer, and mechanical or ultrasonic homogenization at room temperature for 0.5-1 min. Then centrifuge at 13,000g for 15min at 4 ° C. Take the supernatant as a sample.
2. Electrophoresis: prepare an electrophoresis gel and perform SDS-PAGE.
3. Transfer: (Semi-dry transfer)
①After the electrophoresis is finished, cut the rubber strip to the appropriate size, and balance it with the transfer membrane buffer, 5min × 3 times.
â‘¡ Membrane treatment: pre-cut filter paper and NC membrane of the same size as the rubber strip, and immerse in the membrane buffer for 10min.
â‘¢Film transfer: The membrane transfer device is placed in order from the anode carbon plate, 24 layers of filter paper, NC membrane, gel, 24 layers of filter paper, and cathode carbon plate in order from bottom to top. The filter paper, gel, and NC membrane are precisely aligned, each step Remove air bubbles, press up on 500g weight, and absorb excess liquid on the carbon plate. Turn on the power, constant current 1mA / cm2, transfer 1.5hr. After the transfer is completed, disconnect the power and take out the membrane. Cut the membrane strip to be tested for immunoblotting. Dye the band with protein standard, put it in the membrane staining solution for 50s, and then decolorize it in 50% methanol many times until the background is clear, then wash it with double distilled water, air dry and store it in two layers of filter paper. Compare the results.
4. Immune response:
1. Wash the membrane with 0.01M PBS, 5min × 3 times.
2. Add coating solution and shake steadily at room temperature for 2hr.
3. Discard the coating solution and wash the membrane with 0.01M PBS for 5min × 3 times.
4. Add the primary antibody (diluted with 0.01M PBS according to the appropriate dilution ratio, the liquid must cover all of the membrane), and place at 4 ℃ for more than 12hr. For the negative control, replace the primary antibody with 1% BSA. The remaining steps are the same as the experimental group.
5. Discard the primary antibody and 1% BSA, wash the membrane with 0.01M PBS, 5min × 4 times.
6. Add horseradish peroxidase-conjugated secondary antibody (diluted with 0.01M PBS at the appropriate dilution ratio), shake steadily at room temperature for 2hr.
7. Discard the secondary antibody, wash the membrane with 0.01M PBS, 5min × 4 times.
8. Add color developing solution, avoid color development when light appears, and put into double distilled water to stop the reaction.
Five, matters needing attention:
1. The dilution, action time and temperature of the primary and secondary antibodies should be pre-experimented to determine the optimal conditions for different proteins.
2. The color developing solution must be freshly configured and used, and finally H2O2 is added.
3. DAB has the potential to cause cancer, so be careful when handling.
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