The process of Western blot immunoassay involves the separation of protein bands on the gel onto the nitrocellulose membrane by electrophoresis after gel electrophoresis. After blocking, the antibody against the protein to be detected is used as a probe. The combination, after washing, the membrane is combined with a secondary reagent-radiolabeled or horseradish peroxidase or alkaline phosphatase-conjugated anti-immunoglobulin antibody, further washed, and then autoradiographed or original The enzyme reaction determines the position and abundance of the antigen-antibody-antibody complex on the filter.
[Reagents required for Western blotting experiments]
1.IgG standard
2. Goat anti-human horseradish peroxidase (HRP) labeled IgG antibody
3. Transfer buffer: Tris 3.03g, Gly14.4g, methanol 200ml, add three distilled water to 1000ml fully dissolved, 4 ° C refrigerator storage.
4. Tris buffer (TBS): Tris 2.42g, NaCl 29.2g, dissolved in 600ml of three distilled water, then adjusted to pH 7.5 with 1N HCl, then add three distilled water to 1000ml.
5. Rinsing solution (TTBS): TBS liquid 500ml, plus Tween20 250ul.
6. Blocking solution: 5% skim milk powder.
7. Antibody buffer: 1.5 g BSA dissolved in 50 ml TTBS.
8. Development solution DAB (3.3-diaminobenzidine, 3.3-diaminobenzidine) preparation: 5 mg DAB dissolved in 10 ml citric acid buffer (0.01 mol/L citric acid 2.6 ml, 0.02 mol/L Na2HPO4 17.39 ml), plus 30% H2O2 10
Ll (when used).
9. Decolorization solution: methanol 250ml, glacial acetic acid 100ml, add distilled water to 1000ml.
10. Amino black staining solution (0.1% amino black-10B): 0.2 g of amino black-10B dissolved in 200 ml of decolorizing solution, fully stirred and dissolved, and filtered by filter paper.
[Western blotting experimental procedure]
1. SDS-polyacrylamide gel electrophoresis of samples
According to the experimental four steps. When loading, pay attention to making a repeating sample on the same piece of glue in order to prepare for the end of electrophoresis, one for immunoassay and one for protein staining, to facilitate mutual comparison and analyze the experimental results.
Second, transfer imprint
1. Preparation before transfer: Cut the filter paper and nitrocellulose membrane (NC) into the same size as the glue. The NC membrane was immersed in distilled water for 10-20 min and then immersed in the transfer buffer for 30 min.
2. Gel balance: The SDS-PAGE rubber plate after electrophoresis was placed in a transfer buffer for 30-60 min.
3. According to the figure: flatten layer by layer, do not leave bubbles and wrinkles between the layers.
4. Start the transfer, connect the positive and negative poles, cover the lid, connect the power supply, constant current 0.8mA/cm, transfer at room temperature for 1h, and transfer the gel after dyeing with amino black 10B staining solution for 20min, then decolorize and detect the transfer effect. .
Third, immunostaining
1. The transferred NC membrane was blocked in 5% skim milk powder and left overnight at 4 °C.
2. TBS wash film 1-2 times, 10 min / time.
3. Add HRP-labeled antibody at room temperature for 1 h.
4. TBS was washed 3 times, 10 min/time.
5. The NC membrane was transferred to the DAB coloring solution, and the reaction was carried out in the dark. When the color reaction reached an optimum level, the reaction was immediately stopped by washing with three distilled water.
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