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Western Blot
Substrate chemiluminescence ECL method:
Principle of experimental method:
Western Blot (Western Blot) is a method of transferring proteins to the membrane and then using antibodies for detection. 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.
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.
Experimental materials: protein samples
Reagents and kits: acrylamide SDS Tris-HCl β-mercaptoethanol ddH2O glycine Tris methanol PBS NaCl KCl Na2HPO4 KH2PO4 ddH2O Coomassie blue acetic acid skimmed milk powder nickel amine sulfate H2O2 DAB kit
Instruments, consumables: electrophoresis instrument electrophoresis tank centrifuge centrifuge tube nitrocellulose membrane homogenizer scissors pipette gun scraper
Experimental steps:
1. 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; ddH2O 2.8 ml.
3. Transfer membrane buffer: glycine 2.9 g; Tris 5.8 g; SDS 0.37 g; methanol 200 ml; add ddH2O to bring the volume to 1000 ml.
4. 0.01 M PBS (pH7.4): NaCl 8.0 g; KCl 0.2 g; Na2HPO4 1.44 g; KH2PO4 0.24 g; add ddH2O to 1000 ml.
5. Membrane staining solution: Coomassie brilliant blue 0.2 g; methanol 80 ml; acetic acid 2 ml; ddH2O118 ml. Coating solution (5% skimmed milk powder, now prepared): 1.0 g of skimmed milk powder 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 amine sulfate 0.1 ml; H202 1.0 μl.
Second, protein sample preparation
1. Extraction of total protein from monolayer adherent cells
(1) Pour off the culture fluid, and buckle the bottle upside down on the absorbent paper to make the absorbent paper absorb the culture fluid (or place the bottle upright for a while to let the residual culture fluid flow to the bottom of the bottle and then use a pipette to suck it away) .
(2) Add 3 ml of 4 ° C pre-chilled PBS (0.01M pH7.2 ～ 7.3) to each bottle of cells. Wash the cells by gently shaking for 1 min, and then discard the washing solution. Repeat the above operation twice, and wash the cells three times to wash off the culture solution. After discarding the PBS, place the flask on ice.
(3) Add 10 μl PMSF (100 mM) to 1 ml of lysate, shake and place on ice. (PMSF must be shaken until there is no crystal before mixing with the lysis solution.)
(4) Add 400 μl of PMSF-containing lysis solution to each bottle of cells and lyse them on ice for 30 min. To fully lyse the cells, shake the culture bottle back and forth frequently.
(5) After the lysis, scrape the cells on the side of the culture flask with a clean scraper (fast action), and then use a gun to move the cell debris and lysate to a 1.5 ml centrifuge tube. (The whole operation should be done on ice as much as possible.)
(6) Centrifuge at 12000 rpm for 5 min at 4 ° C. (Open the centrifuge in advance to pre-cool)
(7) Transfer the centrifuged supernatant into 0.5 ml centrifuge tubes and store at -20 ℃.
2. Extraction of total protein from tissue
(1) Place a small amount of tissue block in the spherical part of the 1-2 ml homogenizer, and use clean scissors to cut the tissue block as much as possible.
(2) Add 400 μL of single detergent lysate (containing PMSF) to the homogenizer to homogenize. Then put on ice.
(3) After a few minutes, crush it for a while and then put it on ice. Repeat the crushing several times to crush the tissue as much as possible.
(4) After lysis for 30 min, you can use a pipette to transfer the lysate to a 1.5 ml centrifuge tube, and then centrifuge at 12000 rpm for 5 min at 4 ° C. Take the supernatant into 0.5 ml centrifuge tube and place it in- Store at 20 ℃.
3. Extraction of total protein from adherent cells treated with drugs
Due to the influence of drugs, some cells fall off, so in addition to the operation of 1, cells in the culture medium should be collected. The following is the extraction of total cell protein from the culture medium:
(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 discarding the supernatant, washing with PBS was repeated once.
(3) After washing the supernatant with a gun, add 100 μL of lysis solution (containing PMSF) to lyse on ice for 30 min. During the lysis process, a bomb should be played frequently to fully lyse the cells.
(4) Mix the lysate with the lysate in the culture flask and centrifuge at 4 ° C and 12000 rpm for 5 min. Take the supernatant into 0.5 ml centrifuge tubes and store at -20 ° C.
3. Determination of protein content
1. Make a standard curve
(1) Take out 1 mg / ml BSA from -20 ℃, thaw at room temperature, and set aside.
(2) Take 18 1.5 ml centrifuge tubes, each in groups of 3, 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 according to the following table.
(4) After mixing, leave at room temperature for 2 min. Colorimetric analysis on a Bio-Photometer (Eppendorf).
2. Test the protein content of the sample
(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 being left at room temperature for 30 min, 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 a cuvette and measure the blank by blank under the procedure of making a standard curve sample.
(3) Discard the blank sample, wash the cuvette twice with absolute ethanol (0.5 mL each time), and wash again with sterile water.
(4) Take a tube of Coomassie Brilliant Blue plus 95 ml of 0.15 mol / L NaCl NaCl solution and 5 ml of protein sample to be tested, mix it and let it stand for 2 min, pour it into a buttoned cuvette and press the sample button to measure the sample.
Note: For each sample, wash the cuvette twice with absolute ethanol and once with sterile water. Multiple samples can be mixed at the same time and then tested together, which can save a lot of time for measuring a large number of protein samples. The measured result is the amount of protein in the 5 ml sample.
4. SDS-PAGE electrophoresis
1. Clean the glass plate
Fasten the glass plate with one hand and gently scrub with the other hand with a little detergent. After scrubbing both sides, rinse with tap water, rinse with distilled water and stand in a basket to dry.
2. Pouring and loading
(1) After aligning the glass plates, put them in the clips and tighten. Then vertically stuck on the shelf to prepare the glue. (Align the two glasses during operation to avoid leakage.)
(2) Mix the 10% separation gel according to the previous method. After adding TEMED, shake immediately to fill the gel. When pouring glue, you can use a 10 ml gun to suck 5 ml of glue and release it along the glass, when the glue surface rises to the height of the middle line of the green belt. Then add a layer of water on the glue, the gel after the liquid seal is faster. (It can start faster when pouring glue, and slow down when the glue surface reaches the desired height. The glue must flow down the glass plate during operation, so that there will be no bubbles in the glue. When adding water, seal slowly. The glue will be deformed.)
(3) When there is a refraction line between the water and the glue, it means that the glue has set. Wait 3 minutes to allow the glue to fully solidify, then pour off the upper layer of water and absorb the water with absorbent paper.
(4) Mix 4% concentrated gel according to the previous method. After adding TEMED, shake immediately to fill the gel. Fill the remaining space with concentrated gel and insert the comb into the concentrated gel. When pouring 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 when the gel is solidified, which reduces the loading volume of the sample hole, it is often necessary to fill the gel on both sides during the solidification of the concentrated gel. After the concentrated gel has solidified, hold the sides of the comb with both hands and gently pull it up.
(5) Rinse the concentrated gel with water and place it in the electrophoresis tank. (The small glass plate faces inwards, and the large glass plate faces outwards. If only one piece of glue is to be run, a plastic plate should be placed on the other side of the slot and the side with the words facing outwards.)
(6) After measuring the protein content, calculate the volume of the solution containing 50 ng of protein as the sample volume. Take the loading sample into a 0.5 ml centrifuge tube and add 5 × SDS loading buffer to a final concentration of 1 ×. (The total volume of the sample is generally not more than 15 μl, and the maximum sample volume can be 20 μl.) The sample should be boiled in boiling water for 5 min to denature the protein before loading.
(7) After adding enough electrophoresis solution, start to prepare for loading. (At least the electrophoresis liquid should pass through the small glass plate for internal measurement.) Use the micro sampler to suck the sample against the wall, and suck the sample out without air bubbles. Insert the sampler needle into the sample hole and slowly add the sample. (Sampling too fast can cause the sample to flush out of the sample hole, and if there are bubbles, it may overflow the sample. When adding the next sample, the sampler needs to be washed 3 times in the outer tank electrophoresis buffer to avoid cross-contamination.
3. Electrophoresis
The electrophoresis time is generally 4 ~ 5 h, the voltage is preferably 40 V, 60 V can also be used. After electrophoresis, bromophenol blue can be stopped immediately after running out, and the membrane can be transferred.
Five, transfer film
1. To transfer one membrane, prepare 6 sheets of 7.0-8.3 cm filter paper and 1 sheet of nitrocellulose membrane of 7.3-8.6 cm. Be sure to wear gloves when cutting the filter paper and membrane, because the protein on your hands will contaminate the membrane. Put the cut nitrocellulose membrane on the water for 2 h before use. (Use tweezers to hold one side of the membrane gently and place it in a plate with ultrapure water. To float the membrane on the water, only the lower layer is in contact with the water. This will wet the entire membrane due to capillary action. If the membrane sinks into water Here, an air film is formed between the membrane and the water, which prevents the membrane from absorbing water.
2. Put the clips for membrane transfer, two sponge pads, a glass rod, filter paper and the soaked membrane 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 roll back and forth several times to remove the bubbles inside. (Roll with the other hand to hold down the mat so that it cannot move casually.) Put three layers of filter paper on the mat (three sheets of paper can be stacked on the mat first), fix the filter paper in one hand, and use a glass rod to roll out one of them. bubble.
4. Pry off the glass plate before peeling off the glue. When prying, move gently, and pry gently on both sides. After prying for a while, the glass starts to loosen until the glass is pryed off. (Be careful when prying, the glass plate is easy to crack.) After removing the small glass plate, gently scrape the concentrated gel (the concentrated gel affects the operation), to avoid scratching the separating gel. Carefully peel off the separation gel on the filter paper, adjust it by hand to align with the filter paper, and gently roll out the bubbles with a glass rod. Cover the membrane with glue, covering the entire glue (cannot be moved after the membrane cover) and remove air bubbles. Cover the membrane with 3 sheets of filter paper and remove air bubbles. Finally, cover with another sponge pad and roll it together to close the clip. The whole operation is carried out in the transfer liquid, and the air bubbles should be rolled out continuously. The filter paper on both sides of the membrane should not be in contact with each other, and a short circuit will occur after contact. (The transfer fluid contains methanol. Wear gloves when handling. The laboratory must open the door to allow air to circulate.)
5. Put the clip into the groove of the transfer slot, so that the black side of the clip faces the black side of the groove and the white side of the clip faces the red side of the groove. Heat is generated during electric transfer, and a piece of ice is placed on the side of the tank to cool down. Generally use 60 V to transfer for 2 h or 40 V to transfer for 3 h.
6. After the transfer, the membrane was stained with 1 × Ponceau Stain for 5 min (shaking on a decolorizing shaker). Then rinse off the unstained dye with water to see the protein on the membrane. Allow the film to dry for use.
6. Immune response
1. After immersing the membrane in TBS from bottom to top, move to a dish containing the blocking solution, and shake at a room temperature for 1 hour in a decolorizing shaker.
2. Dilute the primary antibody with TBST to the appropriate concentration (in a 1.5 ml centrifuge tube); tear off a piece of cling film of appropriate size and spread it on the experimental table, soak the four corners with water to keep the cling film flat; add the antibody solution Onto the fresh-keeping film; remove the film from the blocking solution, absorb the residual liquid with filter paper, put the membrane protein face down on the antibody liquid surface, tilt the four corners of the membrane to expel the residual air bubbles; after incubation at room temperature for 1-2 hours , Washed twice with TBST on a decolorizing shaker at room temperature for 10 min each time; washed with TBS once again for 10 min.
3. Prepare the secondary antibody dilution and contact with the membrane in the same way as above. After incubating for 1 to 2 hours at room temperature, wash twice with a TBST on a decolorizing shaker at room temperature for 10 minutes each time; wash again with TBS for 10 minutes each time. Chemiluminescence reaction.
7. Chemiluminescence, development, fixing
1. Mix the two reagents A and B in equal volume on the cling film; after 1 min, fully contact the membrane protein face down with this mixed solution; after 1 min, move the film to another cling film and remove The residual liquid is wrapped and placed in the X-ray film holder.
2. In a dark room, put 1 × developer solution and fixer solution into plastic trays separately; take out 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, it cannot be moved, 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 multiple 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, quickly immerse it in the developing solution for development, when obvious bands appear Immediately after that, the development is terminated. The development time is generally 1 to 2 min (20 to 25 ° C). When the temperature is too low (below 16 ° C), the development time needs to be extended appropriately; after the development is completed, the X-ray film is immersed in the fixing solution immediately. 5 to 10 minutes, until the film is transparent; after washing away the remaining fixing solution with tap water, it is allowed to dry at room temperature.
It should be noted that when developing and fixing the film, move the film as much as possible, and do not scratch the film with your fingernail, otherwise it will affect the result.
Eight, gel image analysis
The film was scanned or photographed, and the molecular weight and net optical density values ​​of the target band were analyzed with a gel image processing system.
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