Western blotting

Immunoblotting is the transfer of proteins to a membrane and subsequent detection using antibodies. For the known expressed protein, the corresponding antibody can be used as the primary antibody for detection. The expression product of the new gene can be detected by the fusion part of the antibody, and has the advantages of large analytical capacity, high sensitivity, strong specificity, etc. One of the most commonly used methods of expression and distribution, such as qualitative and quantitative detection of tissue antigens, mass determination of polypeptide molecules, and detection of antibodies or antigens of viruses. Basic Information Specialist classification: growth and development check classification: biochemical examination Applicable gender: whether men and women apply fasting: not fasting Tips: Obey the doctor's instructions. Normal value No special color reaction. Clinical significance Abnormal results: There is a special color reaction, which proves that a certain protein exists. Need to check the crowd: check for a certain protein. Precautions Taboo when checking: None. Taboo when checking: 1. The dilution, action time and temperature of the primary antibody and the secondary antibody are determined by pre-experiment 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 for carcinogenicity, be careful when handling. Inspection process (A) protein sample obtained: after bacterial induction of expression, the cells can be directly lysed by electrophoresis loading buffer, eukaryotic cells plus homogenization buffer, mechanical or ultrasonic room temperature homogenate 0.5-1min. It was then centrifuged at 13,000 g for 15 min at 4 °C. Take the supernatant as a sample. (2) Electrophoresis: An electrophoresis gel was prepared and subjected to SDS-PAGE. (3) Transfer: (semi-dry transfer) 1. After the electrophoresis is finished, cut the strip to the appropriate size and equilibrate with the membrane buffer, 5 min × 3 times. 2. Membrane treatment: The filter paper and NC membrane of the same size as the strip were pre-cut and immersed in the transfer buffer for 10 min. 3. Transfer film: The film transfer device is placed in order from bottom to top according to the order of anode carbon plate, 24 layers of filter paper, NC film, gel, 24 layers of filter paper and cathode carbon plate. The filter paper, gel and NC film are precisely aligned. The bubbles were removed in one step, and a weight of 500 g was applied thereto to dry the excess liquid on the carbon plate. Turn on the power, constant current 1mA/cm2, transfer 1.5hr. After the transfer is completed, the power is removed and the membrane is taken out, and the strip to be tested is cut for immunoblotting. The protein standard strips were stained, placed in the membrane staining solution for 50 s, and then decolorized in 50% methanol several times to a clear background, then washed with double distilled water, air-dried in two layers of filter paper, and left to color The results are compared. (4) Immune response: 1. Wash the membrane with 0.01 M PBS for 5 min x 3 times. 2. Add the coating solution and shake gently at room temperature for 2 hr. 3. Discard the solution and wash the membrane with 0.01 M PBS for 5 min × 3 times. 4. Add primary antibody (diluted with 0.01 M PBS in a suitable dilution ratio, the liquid must cover all of the membrane), and leave at 4 °C for more than 12 hr. In the negative control, the primary antibody was replaced with 1% BSA, and the remaining steps were the same as in the experimental group. 5. Discard the primary antibody and 1% BSA, and wash the membrane with 0.01 M PBS, 5 min × 4 times. 6. Add horseradish peroxidase-conjugated secondary antibody (diluted with 0.01 M PBS at the appropriate dilution ratio) and shake gently for 2 hr at room temperature. 7. Discard the secondary antibody and wash the membrane with 0.01 M PBS for 5 min × 4 times. 8. Add the coloring solution, avoid the light and develop color until the band appears. Put in the double distilled water to stop the reaction. Not suitable for the crowd Not suitable for the crowd: no. Adverse reactions and risks No related complications or hazards.