Experimental principle:
The Western blot method, also known as immunoblotting, separates the protein samples of different molecular weights by SDS-polyacrylamide gel electrophoresis, and transfers the proteins separated on the gel to the solid by transfer electrophoresis. On the phase support (NC membrane or PVDF membrane), the non-labeled antibody against the target protein (primary antibody) is specifically bound to the target protein on the membrane after transfer, and then labeled with horseradish peroxidase. The secondary antibody of the combination is combined and finally detected by the ECL hypersensitive luminescent reagent. If the target protein is contained on the transfer film, after exposure and development by X-ray film, a specific protein band appears on the X-ray film.
Experimental Materials:
Separation gel and laminated glue solution, water-saturated isobutanol, 1×Tris·Cl/SDS, pH8.8, protein molecular weight standard mixture, 1×SDS running buffer, electrophoresis device and clip, glass plate, potting bracket, Buffer tank and other Accessories, 0.75mm edge sealing gasket, 0.75mm sample comb, 50μl micro sampler, constant current power supply
Common reagents:
1) 30% acrylamide / 0.8% N, N'-methylene acrylamide
30 g of acrylamide and 0.8 g of N,N'-methylene acrylamide were dissolved in a total volume of 60 ml of water, heated to 37 ° C to dissolve, and water was added to a final volume of 100 ml. The 0.45 μm microporous membrane was filtered and sterilized, and the pH of the solution was checked to be not more than 7.0, and stored in a brown bottle.
Caution: Acrylamide is highly neurotoxic and can be absorbed through the skin, and its action is cumulative. Gloves and masks should be worn when weighing acrylamide and N,N'-methylene acrylamide. Polyacrylamide is considered to be non-toxic, but care should be taken as it may also contain small amounts of unpolymerized material.
2) 4×Tris·Cl/SDS, pH 8.8,
91 g of Tris base (1.5 mol/L) was dissolved in 300 ml of H2O, the pH was adjusted to 8.8 with 1 mol/L, and H2O was added to a volume of 500 ml. The solution was filtered through a 0.45 μm filter, and 2 g of SDS [0.4% (w/v)] was added thereto, and it was stored at 4 ° C for 1 month.
3) 4×Tris·Cl/SDS, pH 6.8,
6.05 g of Tris base (0.5 mol/L) was dissolved in 40 ml of H2O, the pH was adjusted to 6.8 with 1 mol/L, and H2O was added to a volume of 100 ml. The solution was filtered through a 0.45 μm filter, and 0.4 g of SDS [0.4% (w/v)] was added thereto, and it was stored at 4 ° C for 1 month.
4) 4×SDS running buffer
Tris base 24.2 g , Glycerin 115.3 g , 20% SDS 20 ml
Add water to a total volume of 1000 ml.
When diluted 4 times, it was 1×SDS running buffer (Tris 0.05M, Glycerin 0.38M, SDS 0.1%).
5) TEMED (N, N, N', N'-tetramethylethylenediamine)
TEMED accelerates the polymerization of acrylamide and N,N'-methylene acrylamide by catalyzing the formation of free radicals by ammonium persulfate.
6) 10% ammonium persulfate
Ammonium persulfate provides the free radicals necessary to drive the polymerization of acrylamide and methylene acrylamide. A small amount of 10% (w/v) hydrazine stock solution can be prepared with deionized water and stored at 4 °C. Since ammonium persulfate will slowly decompose, it should be freshly prepared every other week.
Experimental steps:
After transfection, the sample was taken, centrifuged at 12000 rpm for 2 min, and the supernatant was removed. The cell debris was directly subjected to the following steps:
(1) Add 10 μl of RIPA buffer/200 μl per well and lyse for 30 min at 4°.
(2) Prepare for thermal denaturation, using an 8-tube tube, add 5 x loading buffer 5 μl per well.
(3) Using a 20 μl pipette, take 20 μl of the lysed cells, add to the corresponding eight tubes, and mix by pipetting.
(4) Sealing, heat denaturation treatment with a PCR machine: 97 ° 8 min.
(5) Store the denatured sample at 4° for testing.
1. Preparation of SDS-PAGE gel
The mixture was pre-formulated (Tris-HCL/Acr-bis/SDS/H2O), and APS and TEMED were added at the time of gel preparation.
Separation gel: 1.5 M Tris-HCL 8.8, 10% separation gel. (The so-called 10% concentration is the concentration of Acr-Bis, ie the concentration of acrylamide, the volume is fixed with water, the other components are unchanged); the concentrated gel: using 1M Tirs-HCL 6.8, concentration: 5%.
2, loading electrophoresis:
The comb used is 15 holes, and the Marker occupies one hole, so 14 samples can be detected per gel. Load: Marker: 5 μl/well; Sample: 20 μl/well; Parameter: 80 V, wait for the sample to run out of the sample well; 120 V, the sample enters the separation gel; 200 V, until the end (blue indication is brought to the bottom of the gel Complete electrophoresis)
3, transfer film:
Transfer film sandwich structure order: black plate, mesh pad, a layer of filter paper, gel (used to put Marker on the right side), NC film (marked in the upper left corner of the protein surface of the film), two layers of filter paper, mesh mat, White plate.
Remarks: The filter paper used is also divided into a rough surface and a fine surface, which is delicately facing the gel and the NC film, and is rough facing the mesh mat. When the clip of the sandwich structure is placed in the electrode, the black plate corresponds to the black side (negative electrode) of the electrode and the red side (positive electrode) of the corresponding electrode of the white plate.
It is emphasized that the order of the gel and the NC film (nitrocellulose membrane) should not be reversed. The NC membrane used is divided into a rough surface and a smooth surface, and the rough surface is a protein-receiving surface which is in direct contact with the gel. When covering the slot cover, also note that black corresponds to black and red corresponds to red; when the wire electrode is connected to the power supply, it is also black to black, red to red. Any of the above steps will lead to protein transfer failure, remember.
Parameters used for the transfer: steady flow 45 mA, overnight (999 min).
4, closed
The blocking solution used: 5% Nonfat milk, prepared with TBST, RT (room temperature), 30 min~1 h (determined according to the actual situation, such as the number of times the blocking solution has been used, the background of ECL color development). In order to prevent the deterioration of milk powder, add sodium azide, NaN3 (a kind of spectral preservative), to a final concentration of 0.02% (the concentration of sodium azide in the laboratory is 2%)
Wash: TBST, wash away the residual milk powder.
5, primary antibody incubation
Antibody used: mouse, Anti-Flag.
Primary antibody dilution: 4% BSA by Super Block, 1:1000. 0.02% sodium azide NaN3 was also added.
Incubation: RT, approximately 3 h.
Wash: TBST, 3 x 5min
6, secondary antibody incubation
Antibody used: HRP, anti-mouse.
Secondary antibody dilution: 5% Nonfat milk by TBST, 1:2000. (The secondary antibody is generally renewed twice or three times) (emphasis: NaN3 may not be added to the secondary antibody because it inhibits HRP activity)
Incubation: RT, 1 h.
Wash: TBST, 6 x 5min.
7, color: ECL.
A liquid / B liquid, each is taken in an equal volume of 300 μl, and is currently used. Mark the Marker on the NC film and the position of the sample well with a highlighter. The reaction solution was uniformly applied to the membrane (protein surface), the whole membrane was wetted, and then the protein of the membrane was placed face down on the colorimeter, the lid was closed, and the start was clicked.
Precautions
1. The board that starts to be glued must be clamped to prevent leakage.
2, the sample board has to be clamped to prevent leakage of liquid and slow liquid, the speed of the glue in the running rubber is not the same, the reason is that the two pieces of glue before the board is not tightened the leakage of liquid, and the concentration of the glue is not the same.
3, when loading, marker5ul plus 5 x loading buffer mix and load together, this can prevent the process of running the electrophoresis, the sample is getting narrower and narrower.
4. When transferring the film, the upper left corner of the front surface of the NC film is marked with a marker pen to prevent the process of incubation of the antibody and color development from being able to distinguish the front and back sides of the NC film.
5, before the film transfer, first put the NC film in the transfer film soak, pay attention to slowly soak from one end down, otherwise the NC film is easy to bubble, affecting the film.
6. When coloring, add the film transfer liquid to the NC film and slowly add it from one side to the bottom. Otherwise, the final color development effect is not good.
7. When transferring film, there must be no air bubbles before the contact between the NC film and the glue. Otherwise, it has an effect on the purpose of the film.
common problem
1. The film is a blank, what is going on?
If the following problems can be ruled out, then the problem usually occurs in the primary antibody and antigen preparation.
1) The HRP activity of the secondary antibody is too strong to consume the substrate;
2) H2O2 in the ECL substrate is unstable and inactivated;
3) The ECL substrate is not covered to the corresponding position;
4) Improper selection of primary antibodies;
5) The secondary antibody is inactivated.
2, the background is high?
The film is not completely evenly wetted, it is recommended to use 100% methanol saturated film;
Insufficient film washing can increase the volume of washing liquid and the number of washings;
The electrode is unbalanced or the loading position is skewed, and the electrode and the sample can be adjusted;
Insufficient amount of the closure can increase the concentration of the closure, and ensure that the blocking solution is completely immersed in the transfer film during incubation;
Improper use of the closure, such as detection of biotinylated proteins, may not be blocked with skimmed milk powder;
The sealing time is not enough. Under normal circumstances, the room temperature is closed at 37 degrees for more than 1 hour or closed at 4 degrees overnight;
7) Non-specific binding of antibodies can reduce antibody concentration and reduce incubation time
8) The dilution of the primary antibody is not suitable. The antibody can be titrated to select the most suitable antibody dilution.
9) The temperature of primary antibody incubation is high, it is recommended to combine overnight at 4 °C.
10) The antibody concentration is too high or insufficiently washed. It is recommended to reduce the antibody concentration and increase the number of washings and time.
11) Too many chemical coloring substrates, it is recommended to add an appropriate amount of chromogenic substrate according to the instructions.
3, the shape of the strip is not good?
1) The unevenness of the gelation or the polymerization is not good. It is recommended to mix the solution thoroughly before filling the glue.
2) Some samples have higher salt concentration, it is recommended to remove salt or adjust the salt concentration of the sample to be consistent.
3) The buffer is old, the composition is changed, and it can be reconfigured.
4) There are bubbles under the gel, and the bubbles should be removed before electrophoresis.
5) When the temperature is too high during electrophoresis, the current or voltage can be reduced.
6) The sample contains insoluble particles. It is recommended that the sample be thoroughly stirred and mixed.
4. Is the protein strip position (size) incorrect?
The concentration of the glue is not correct. The position of the protein bands run by different concentrations of glue may be biased. The concentration of the antibody may be adjusted. The incubation of the antibody is insufficient. It is recommended to increase the antibody concentration and prolong the incubation time.
3) Inactivation of the enzyme, it is recommended to directly mix the enzyme and the substrate. If the color is not developed, the enzyme is inactivated. Select active enzymes during the period of validity
4) There are post-translational modifications or splicing of the target protein.
5) The target protein or target protein content in the specimen is too low. It is recommended to set the positive control comparison result and increase the specimen loading.
5. Is there a lot of miscellaneous things?
1) The target protein has multiple modification sites, and it can present multiple bands by itself. It is recommended to consult the literature or perform bioinformatics analysis to obtain the modification site information of the protein sequence, and determine the actual size of the protein by de-modification.
2) Degradation of the target protein during sample processing, it is recommended to add protease inhibitor; operate on ice during sample processing
3) There are many heteroproteins, and it is recommended to treat the protein of interest.
4) The antibody specificity is not strong, it is recommended to use a specific antibody
5) Antibody incubation time is too long, it is recommended to reduce antibody incubation time
6) The secondary antibody cross-reacts with the antigen, it is recommended to choose a suitable closure.
7) Dimer or multimer is present, it is recommended to increase protein denaturation process and strength
8) Substrate color development and exposure time is too long, it is recommended to shorten the color development and exposure time
6, the background has black spots or uneven white spots and white bands on a dark background
1) The antibody reacts with the blocking reagent, it is recommended to filter the blocking reagent before use.
2) HRP content is too high, it is recommended to reduce the concentration of enzyme-linked secondary antibodies
3) There are aggregates in the HRP-coupled secondary antibody, it is recommended to filter the secondary antibody to remove aggregates.
4) The antibody is not evenly distributed. It is recommended to use a shaker when incubating the antibody.
7, the cell level to do WB, how many cells raised enough to do WB?
A: Generally 5×10^6 is enough.
8. Why is the target protein not detected in my cell extract?
Possible reasons are:
1) The protein is not expressed in the cell, and the cell is replaced by a cell;
2) The antibody does not recognize the target protein, so look at the instructions and see if there is a problem;
3) It may not be kept at low temperature, the sample is not properly stored, and the sample is placed for too long;
4) The protein in the cell is degraded by the enzyme, and a protease inhibitor can be added to inhibit the protease activity.
9. My purpose is very weak. How to strengthen it?
1) It is possible to increase the amount of antigen loaded, which is the most important;
2) It is also possible to reduce the dilution ratio of the primary antibody;
3) It is also possible to extend the exposure time.
10. The molecular weight of the protein I made is very small (10KD). How do I do WB?
1) PVDF film or NC film with a pore size of 0.22 um can be selected, the film transfer time is shortened, and a Tricine-SDS-PAGE system can be used;
2) The PSQ film can also be selected while reducing the transfer time. It is also possible to stack the two films together and transfer them. Other steps can be used.
11, large molecular weight protein 200KD, what should you pay attention to when doing WB?
1) When making WB of 200kd protein, it should be noted that the separation glue should be selected >7%; be careful when stripping the glue;
2) The transfer time needs to be extended accordingly; the molecular weight reference should be made (otherwise, the miscellaneous band does not know how to analyze);
3) The methanol content in the transfer film can be appropriately reduced. It is recommended to use a wet film transfer film with higher efficiency!
12. If the sample load is overloaded, what method should I use to increase the sample load?
The sample can be concentrated, and a portion of the small molecule protein can also be dialyzed according to the target molecular weight. In general, overloading 30% is not a problem. If you have already exceeded a lot, and the small molecular weight should also be considered, you can consider increasing the thickness of the glue, you can try 1.5mm.
13. Can antibodies in WB be used repeatedly?
Antibody working solutions generally do not claim storage for repeated use, but if the antibody is more precious, it can be used 2-3 times. It should be used within 2-3 days after dilution and stored at 4 degrees to avoid repeated freezing and thawing.
14. What are the requirements for the upper and lower tank buffers? How can I achieve the best results?
No special requirements. However, it is common to put a fresh buffer in the upper tank, and the lower tank may be a buffer that is used once or twice.
15. Can immunohistochemistry and WB use the same antibody?
In immunohistochemistry, antibodies recognize antigenic determinants (also known as epitopes) that have not been denatured, some epitopes are linear, and some are conformational; linear epitopes are not affected by protein denaturation, natural proteins and The boiled protein is contained; the conformational epitope is limited by the spatial structure of the protein, and the denaturing will disappear after cooking. If the antibody used recognizes several amino acids in a protein, that is, a linear epitope, then the antibody can be used for both immunohistochemistry and WB, and if the antibody recognizes a conformational epitope, it can only be used for immunization. Grouped.
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