Western Blot can: (1) detect the target protein from the protein mixture; (2) quantitatively or qualitatively determine the expression of the protein in the cell or tissue; (3) use it for protein-protein, protein-DNA, Subsequent analysis of protein-RNA interactions.
experimental method
- Substrate chemiluminescence ECL method
- Western blotting
- Graphic
| Principle of experimental method | Western Blot is a method in which proteins are transferred to a membrane and then detected using an antibody. For known expression proteins, the corresponding antibody can be used as a primary antibody for detection, and the expression product of the new gene can be detected by the fusion portion of the antibody. |
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| Experimental Materials | Protein sample |
| Reagents, kits | Acrylamide SDS Tris-HCl β-mercaptoethanol ddH2O Glycine Tris Methanol PBS NaCl KCl Na2HPO4 KH2PO4 ddH2O Coomassie brilliant acetic acid skim milk powder nickel sulphate H2O2 DAB kit |
| Instruments, consumables | Electrophoresis electrophoresis tank centrifuge centrifuge tube nitrocellulose membrane homogenizer scissors pipette gun scraper |
| Experimental procedure | First, reagent preparation 1. SDS-PAGE reagent: See polyacrylamide gel electrophoresis experiment. 2. Homogenization buffer: 1.0 M Tris-HCl (pH 6.8) 1.0 ml; 10% SDS 6.0 ml; β-mercaptoethanol 0.2 ml; ddH 2 O 2.8 ml. 3. Transmembrane buffer: glycine 2.9 g; Tris 5.8 g; SDS 0.37 g; methanol 200 ml; add ddH 2 O to 1000 ml. 4. 0.01 M PBS (pH 7.4): NaCl 8.0 g; KCl 0.2 g; Na 2 HPO 4 1.44 g; KH 2 PO 4 0.24 g; add ddH 2 O to 1000 ml. 5. Membrane staining solution: Coomassie brilliant blue 0.2 g; methanol 80 ml; acetic acid 2 ml; ddH 2 O118 ml. Coating solution (5% skimmed milk powder, ready to use): Skim milk powder 1.0 g was dissolved in 20 ml of 0.01 M PBS. 6. Color developing solution: DAB 6.0 mg; 0.01 M PBS 10.0 ml; nickel sulfate amine 0.1 ml; H 2 0 2 1.0 μl. Second, protein sample preparation 1. Extraction of total protein from monolayer adherent cells (1) Pour off the culture solution and pour the bottle onto the absorbent paper so that the absorbent paper absorbs the culture solution (or place the bottle upright for a while to allow the residual culture solution to flow to the bottom of the bottle and then pipette it away) . (2) Add 3 ml of 4 ° C pre-cooled PBS (0.01 M pH 7.2-7.3) to each vial of cells. Wash the cells gently by shaking gently for 1 min, then discard the wash solution. The above operation was repeated twice, and the cells were washed three times to wash away the culture solution. After PBS was discarded, the flask was placed on ice. (3) Add 1 μl of lysate plus 10 μl of PMSF (100 mM) and shake well on ice. (PMSF should be shaken until no crystals are mixed with the lysate.) (4) Add 400 μl of PMSF-containing lysate to each bottle of cells and lyse on ice for 30 min. In order to fully lyse the cells, shake the flask frequently. (5) After the lysis, scrape the cells on one side of the flask with a clean scraper (faster), then use a gun to transfer the cell debris and lysate to a 1.5 ml centrifuge tube. (The whole operation is carried out on ice as much as possible.) (6) Centrifuge at 12000 rpm for 5 min at 4 °C. (pre-opening centrifuge pre-cooling) (7) Transfer the supernatant from the centrifuge to a 0.5 ml centrifuge tube and store at -20 °C. 2. Extraction of total protein from tissues (1) Place a small amount of tissue block in the globular part of the 1-2 ml homogenizer, and cut the tissue block as much as possible with clean scissors. (2) Add 400 μL of single detergent lysate (containing PMSF) to the homogenizer for homogenization. Then placed on ice. (3) After a few minutes, grind it for a while and then place it on ice. Repeat the grinding several times to make the tissue crush as much as possible. (4) After lysing for 30 min, the lysate can be transferred to a 1.5 ml centrifuge tube with a pipette, then centrifuged at 12000 rpm for 5 min at 4 ° C, and the supernatant is placed in a 0.5 ml centrifuge tube and placed in - Store at 20 ° C. 3. Extraction of total protein from adherent cells treated with drug Since some cells are detached due to the influence of the drug, the cells in the culture solution should be collected in addition to the operation of 1. The following is the extraction of total cellular protein from the culture: (1) Pour the culture solution into a 15 ml centrifuge tube and centrifuge at 2500 rpm for 5 min. (2) Discard the supernatant, add 4 ml of PBS and gently wash with a gun, then centrifuge at 2500 rpm for 5 min. After the supernatant was discarded, it was washed once with PBS. (3) After washing the supernatant with a gun, add 100 μL of lysate (containing PMSF) to lyse on ice for 30 min. During the lysis process, a bomb is often used to fully lyse the cells. (4) The lysate was mixed with the lysate in the culture flask and centrifuged at 12000 rpm for 5 min at 4 ° C. The supernatant was dispensed into a 0.5 ml centrifuge tube and stored at -20 ° C. Third, the determination of protein content Make a standard curve (1) Take 1 mg/ml BSA from -20 °C, melt at room temperature, and set aside. (2) Take 18 1.5 ml centrifuge tubes, 3 groups, labeled 0 mg, 2.5 mg, 5.0 mg, 10.0 mg, 20.0 mg, 40.0 mg. (3) Add various reagents to each tube as shown in the following table. (4) After mixing, leave at room temperature for 2 min. Colorimetric analysis was performed on a biospectrometer (Bio-Photometer, Eppendorf). 2. Detect sample protein content (1) Take a sufficient amount of 1.5 ml centrifuge tubes and add 1 ml of Coomassie Brilliant Blue solution stored at 4 °C to each tube. After 30 minutes at room temperature, it can be used to measure protein. (2) Take a tube of Coomassie Brilliant Blue plus 0.15 mol/L NaCl solution 100 ml, mix and place for 2 minutes to make a blank sample, pour the blank into the cuvette and press blank to measure the blank under the procedure of the standard curve. sample. (3) Discard the blank sample, wash the cuvette twice with absolute ethanol (0.5 mL each time), and then wash it once with sterile water. (4) Take a tube of Coomassie Brilliant Blue plus 95 ml 0.15 mol/L NaCl NaCl solution and 5 ml of the sample protein to be tested, mix and let stand for 2 min, pour into the crusted cuvette and press sample to test the sample. Note: For each sample, the cuvette should be washed twice with absolute ethanol and washed once with sterile water. Multiple samples can be mixed at the same time and tested together, which saves a lot of time for measuring large amounts of protein samples. The measured result is the amount of protein contained in the 5 ml sample. Fourth, SDS-PAGE electrophoresis Cleaning the glass Fasten the glass with one hand and gently scrub with the other hand. After scrubbing on both sides, rinse with tap water, rinse with distilled water, and stand in the basket to dry. 2. Glue and load (1) After aligning the glass plates, put them into the clips and clamp them. Then vertically clamp on the shelf to prepare the glue. (Align the two glasses during operation to avoid leakage.) (2) According to the previous method, 10% separation gel, immediately after adding TEMED, can be filled. When filling the glue, 5 ml of glue can be used to draw 5 ml of glue along the glass, and the glue surface can be raised to the height of the middle line of the green belt. Then add a layer of water to the gel, and the gelation after liquid sealing is faster. (It can start faster when filling the glue, and slow down when the rubber surface reaches the required height. The glue must flow down the glass plate during operation, so there will be no bubbles in the glue. It is very slow when adding the water seal, otherwise The glue will be modified.) (3) When there is a refracting line between the water and the glue, it means that the glue has condensed. Wait another 3 minutes for the glue to solidify enough to pour off the top layer of water and blot the water with absorbent paper. (4) According to the previous method, 4% concentrated glue is added, and immediately after adding TEMED, it can be filled by shaking. Fill the remaining space with the concentrated gel and insert the comb into the concentrate. When filling the glue, the glue should also flow down along the glass plate to avoid bubbles in the glue. Keep the comb level when inserting the comb. Since the volume shrinks and shrinks when the glue solidifies, the loading volume of the sample hole is reduced, so the glue is often applied on both sides during the solidification process of the concentrated glue. After the gel has solidified, pinch the sides of the comb and pull them straight up. (5) Rinse the concentrated gel with water and place it in the electrophoresis tank. (Small glass plate faces inward, large glass plate faces outward. If only one piece of glue is run, the other side of the groove should be padded with a plastic plate and the word side faces outward.) (6) After measuring the protein content, the volume of the solution containing 50 ng of protein is calculated. The sample was taken out into a 0.5 ml centrifuge tube and 5 x SDS loading buffer was added to a final concentration of 1×. (The total volume of the sample is generally not more than 15 μl, and the maximum of the sample hole can be added with 20 μl of the sample.) Before the sample is loaded, the sample is boiled in boiling water for 5 min to denature the protein. (7) Add enough electrophoresis fluid and start preparing for loading. (The electrophoresis fluid should at least flow through the small glass plate that is internally tested.) Dip the sample with a micro-injector and aspirate the sample without inhaling air bubbles. Insert the syringe needle into the well and slowly add the sample. (Sampling too fast can make the sample out of the sample well, if there is air bubbles, it may overflow the sample. When adding the next sample, the injector should be washed 3 times in the outer tank running buffer to avoid cross-contamination. 3. Electrophoresis The electrophoresis time is generally 4 to 5 h, the voltage is 40 V, and 60 V is also available. Electrophoresis until the bromophenol blue has just run out can terminate the electrophoresis and transfer the film. Five, transfer film 1. To transfer a film, prepare 6 sheets of 7.0-8.3 cm filter paper and 1 7.3-8.6 cm nitrocellulose membrane. Always wear gloves when cutting the filter paper and film, as the protein on your hands can contaminate the film. The cut nitrocellulose membrane was immersed in water for 2 h before use. (Pinch the side of the membrane with tweezers and gently place it in a plate with ultrapure water. The membrane should float on the water, only the lower layer will be in contact with water. This will soak the entire membrane due to capillary action. If the membrane sinks into the water Inside, a film of air is formed between the membrane and the water, which prevents the membrane from absorbing water. 2. Place a clip for the transfer film, two sponge pads, a glass rod, filter paper, and a dipped film in the enamel pan with the transfer solution. 3. Open the clip to keep the black side level. Put a sponge pad on it and use a glass rod to rub it back and forth several times to get rid of the bubbles inside. (One hand rubs the other hand to hold the mat so that it can't be moved casually.) Put three layers of filter paper on the mat (three sheets of paper can be stacked on the mat first), and fix the filter paper with one hand and use a glass stick to remove it. bubble. 4. You must first remove the glass plate to peel off the glue. When you are licking, the action should be light. You should gently rub it on both sides. After a while, the glass plate began to loosen until the glass plate was removed. (Be careful when you are careful, the glass plate is very easy to crack.) After removing the small glass plate, gently scrape off the concentrated glue (the concentrated glue affects the operation), and avoid scraping the separation rubber. Carefully peel off the separation rubber cover on the filter paper, adjust it by hand to align it with the filter paper, and gently remove the air bubbles with a glass rod. Cover the film on the glue, cover the entire glue (no more movement after the film cover) and remove the air bubbles. Three sheets of filter paper were placed on the membrane and air bubbles were removed. Finally, cover another sponge pad and fold the clip a few times. The entire operation is carried out in the transfer liquid, and the bubbles are continuously removed. The filter paper on both sides of the film cannot touch each other, and a short circuit occurs after contact. (The transfer liquid contains methanol, gloves should be worn during operation, and the laboratory should open the door to allow air to circulate.) 5. Place the clip into the transfer slot so that the black side of the clip faces the black side of the slot and the white side of the clip faces the red side of the slot. Heat is generated during electrical transfer, and a piece of ice is placed on one side of the tank to cool down. It is generally transferred with 60 V for 2 h or 40 V for 3 h. 6. After the transfer, the membrane was stained with 1× Lichunhong dye for 5 min (shake on a bleaching shaker). The protein on the membrane can then be seen by rinsing off the stained solution with water. Allow the film to dry for use. Sixth, immune response 1. Soak the membrane from the bottom up with TBS, transfer to a dish containing the blocking solution, and shake for 1 h at room temperature on a decolorizing shaker. 2. Dilute the primary antibody to the appropriate concentration with TBST (in a 1.5 ml centrifuge tube); remove the appropriate size of the plastic wrap on the test bench, soak the water in the four corners to keep the wrap film flat; add the antibody solution On the wrap film; remove the film from the blocking solution, and use the filter paper to remove the residual liquid, then place the membrane protein face down on the surface of the antibody, lick the four corners of the membrane to drive out the residual bubbles; incubate for 1 to 2 hours at room temperature Wash twice with TBST on a bleaching shaker at room temperature for 10 min each time; then wash once with TBS for 10 min. 3. Prepare the secondary antibody dilution solution and contact with the membrane as in the above method. Incubate for 1 to 2 hours at room temperature, then wash twice with TBST at room temperature on a decolorizing shaker for 10 min each time; then wash once with TBS for 10 min. Chemiluminescence reaction. Seven, chemiluminescence, development, fixing 1. Mix the two reagents A and B in equal volume on the plastic wrap; after 1 min, bring the membrane protein face down with the mixture; after 1 min, transfer the membrane to another plastic wrap. The residual liquid, wrapped, placed in an X-ray clip. 2. In the dark room, separate the 1× developer and fixing solution into the plastic tray; take the X-ray film under the red light and cut it with a paper cutter to the appropriate size (1 cm larger than the length and width of the film) ); open the X-ray film clip, put the X-ray film on the film, once placed, can not move, close the X-ray film clip, start timing; adjust the exposure time according to the strength of the signal, generally 1 Min or 5 min, you can also choose to press the film several times at different times to achieve the best effect; after the exposure is completed, open the X-ray film clip, take out the X-ray film, and quickly immerse it in the developing solution to develop obvious bands. Immediately, the development is terminated. The development time is generally 1 to 2 min (20 to 25 ° C). When the temperature is too low (less than 16 ° C), the development time should be appropriately extended. Immediately after the development, the X-ray film is immersed in the fixing solution, and the fixing time is generally 5 to 10 minutes, until the film is transparent; wash off the residual fixing solution with tap water, and then dry at room temperature. It should be noted that when developing and fixing the film, try to take a corner of the film, and the fingernails should not scratch the film, otherwise the result will be affected. Eight, gel image analysis The film is scanned or photographed, and the molecular weight and net optical density values ​​of the target tape are analyzed using a gel image processing system. |
| Precautions | 1. The dilution, duration of action and temperature of the primary and secondary antibodies are determined by pre-experimentation for different proteins. 2. The color developing solution must be freshly configured and finally added with H 2 O 2 . 3. DAB has the potential to cause cancer, so be careful when handling it. |
| other | First, the immune response 1. Wash the membrane with 0.01 M PBS for 5 min × 3 times. 2. Add the coating solution and shake gently for 2 h at room temperature. |
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