First, the principle of experimental methods

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, and the expression product of the new gene can be detected by the fusion portion of the antibody.

Similar to the Southern or Northern hybridization method, but Western Blot uses polyacrylamide gel electrophoresis, the detected substance is a protein, the "probe" is an antibody, and the "developing color" uses a labeled secondary antibody.

The protein sample separated by PAGE is transferred to a solid phase carrier (for example, a nitrocellulose membrane), and the solid phase carrier adsorbs the protein as a non-covalent bond, and can maintain the type of the polypeptide separated by electrophoresis and its biological activity. The protein or polypeptide on the solid phase carrier is used as an antigen, and the corresponding antibody is immunoreacted, and then reacted with an enzyme or an isotope-labeled secondary antibody, and the specific purpose of electrophoretic separation is detected by substrate color development or autoradiography. Protein component of gene expression. This technique is also widely used to detect expression at the protein level.

Second, the experimental materials

Protein sample

Third, 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

Fourth, instruments, supplies

Electrophoresis electrophoresis tank centrifuge centrifuge tube nitrocellulose membrane homogenizer scissors pipette gun scraper

Fifth, the experimental steps

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. Transmembrane buffer: glycine 2.9 g; Tris 5.8 g; SDS 0.37 g; methanol 200 ml; add ddH2O to 1000 ml.

4. 0.01 M PBS (pH 7.4): NaCl 8.0 g; KCl 0.2 g; Na2HPO4 1.44 g; KH2PO 40.24 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, 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; H202 1.0 μl.

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 medium (or leave 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 (0.01M 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 vial 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 after centrifugation 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 proteins 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.

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 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 measured 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.

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 is added. Immediately after adding TEMED, the mixture can be filled. When filling the glue, 5 ml of glue can be sucked out along the glass with a 10 ml gun, 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, immediately after adding TEMED, can be filled. 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 gel solidifies, the loading volume of the sample hole is reduced, so the glue is often applied on both sides during the solidification of the gel. 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 as the sample loading. The sample was taken out to 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 tested internally.) 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.

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 dish 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 before you can peel 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 (concentration of the glue affects the operation), to avoid scratching the separation rubber. Carefully peel off the separation rubber cover onto 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 close the clip with a few clicks. 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. When electricity is transferred, heat is generated, 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.

immune response

1. Soak the membrane from bottom to top 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 surface, 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 then use the filter paper to remove the residual liquid, then place the membrane protein face down on the surface of the antibody, and shake 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 on a decolorizing shaker at room temperature for 10 min each time; then wash once with TBS for 10 min. Chemiluminescence reaction.

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. Residual liquid, wrapped, placed in an X-ray clip.

2. In the dark room, separate the 1× developer and fixer into the plastic tray; take the X-ray under the red light and cut it with a paper cutter (1 cm larger than the length and width of the film) ); open the X-ray clip, put the X-ray on the film, once placed, can not move, close the X-ray clip, start timing; adjust the exposure time according to the strength of the signal, generally 1 Min or 5 min, you can 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 (below 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; rinse off the residual fixing solution with tap water and let it 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.

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.

Six, matters needing attention

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 to H2O2.

3. DAB has the potential for carcinogenicity and should be handled with care.

other

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.

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 the appropriate dilution ratio, the liquid must cover all of the membrane), and place at 4 °C for more than 12 h. 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 for 5 min × 4 times.

6. Add horseradish peroxidase-conjugated secondary antibody (diluted with 0.01 M PBS in appropriate dilution) and shake gently for 2 h at room temperature.

7. Discard the secondary antibody and wash the membrane with 0.01 M PBS for 5 min x 4 times.

8. Add the coloring solution and avoid the light to develop color until the band appears. Stop the reaction by placing it in double distilled water.

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