Western Blot

Western Blot transfers proteins to the membrane and then detects them with antibodies. 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.

This article introduces the Western Blot technology in detail through the following aspects:

First, principle two, classification
Autoradiography
Ii. Substrate chemiluminescence ECL
Iii. Substrate Fluorescence ECF
Iv. Substrate DAB color III, main reagent IV, main step 5, common problem guide
1. Reference book recommendation
2. Frequently asked questions about samples
3. Antibody
4. Problems with filter paper, glue and film
5.Marker related questions
6. Selection of dyeing
7. Reference question
8. Frequently Asked Questions for Buffer Formulations
9. Conditional exploration
10. Introduction of the method
11. Analysis of results

First, the principle is similar to that of Southern or Northern hybridization, but Western Blot uses polyacrylamide gel electrophoresis, the detected substance is 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 immunoreactive, and then reacted with the enzyme or the isotope-labeled secondary antibody, and the specific purpose of the 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, classification
The methods of western color development mainly include the following:
Autoradiography
Ii. Substrate chemiluminescence ECL
Iii. Substrate Fluorescence ECF
Iv. Substrate DAB coloring is commonly used in the substrate chemiluminescence ECL and substrate DAB color, the same level and experimental conditions are the first method, the current published article usually uses substrate chemiluminescence ECL. As long as you buy a ready-made kit, the operation is relatively simple, the principle is as follows (the secondary antibody is labeled with HRP): the reaction substrate is peroxide + luminol, if it encounters HRP, it emits light, which can expose the film. Wash out the strips.

Third, the main reagent
1, acrylamide and N, N'-methylene bis acrylamide, should be prepared with warm (to facilitate the dissolution of diacrylamide) deionized water containing 29% (w / v) acrylamide and 1% (w /v) N, N'-methylene bis acrylamide stock solution acrylamide 29g, N, N-methylene fork diacrylamide 1g, add H2O to 100ml. Store in a brown bottle and store at 4°C in the dark. Strictly verify that the pH should not exceed 7.0, because the deamination reaction can be photocatalytic or base catalyzed. The period of use must not exceed two months and must be reconstituted every few months. If there is sediment, it can be filtered.
2. Sodium dodecyl sulfate SDS solution: 10% (w/v) 0.1 g SDS, 1 ml H2O deionized water, and stored at room temperature.
3. Separation gel buffer: 1.5 mmol/LTris-HCL (pH 8.8): 18.15 g Tris and 48 ml 1 mol/L HCL were mixed and diluted with water to a final volume of 100 ml. Store at 40C after filtration.

4. Concentrated gel buffer: 0.5 mmol/LTris-HCL (pH 6.8): 6.05 g of Tris was dissolved in 40 ml of H2O, and adjusted to pH 6.8 with about 48 ml of 1 mol/L HCL and diluted with water to a final volume of 100 ml. Store at 40C after filtration. Both buffers must be prepared using Tris base and the pH adjusted with HCL instead of Tris.CL.

5. The TEMED original solution N, N, N'N' tetramethylethylenediamine catalyzes the formation of free radicals by ammonium persulfate to accelerate the polymerization of the two acrylamides. When the pH is too low, the polymerization reaction is suppressed. 10% (w/v) ammonium persulfate solution. Provides the free radicals necessary for the polymerization of two acrylamides. Prepare a few ml of deionized water, and prepare before use.

6. SDS-PAGE loading buffer: pH6.8 0.5mol/L Tris buffer 8ml, glycerin 6.4ml, 10% SDS 12.8ml, mercaptoethanol 3.2ml, 0.05% bromophenol blue 1.6ml, H2O 32ml mixed . Mix with the protein sample in a ratio of 1:1 or 1:2, and mix it in boiling water for 3 min before mixing, usually 20-25 ul, total protein amount 100 μg.

7. Tris-glycine electrophoresis buffer: 30.3 g Tris, 188 g glycine, 10 g SDS, dissolved in distilled water to 1000 ml, and 0.25 mol/L Tris-1.92 mol/L glycine electrode buffer was obtained. Dilute 10 times before use.

8. Transfer buffer: Prepare 1L transfer buffer, weigh 2.9g glycine, 5.8g Tris base, 0.37g SDS, add 200ml methanol, add water to the total amount of 1L.

9. Lichun red dye solution: Lichunhong S 2g Trichloroacetic acid 30g Sulfosalicylic acid 30g Add water to 100ml When using the above solution, dilute 10 times to form Lichunhong S. It should be discarded after use.

10. Skim milk powder 5% (w/v).

11. NaN3 0.02% sodium azide (toxic, gloved), soluble in phosphate buffered saline (PBS).

12. Tris buffered saline solution (TBS): 20 mmol/LTris/HCL (pH 7.5), 500 mmol/L naCl.

13. Tween20 (15) mouse anti-human-MMP-9 (16) mouse anti-human-TIMP-1.

14. A peroxidase-labeled secondary antibody.

15. NBT (dissolved in 70% dimethylformamide, 75 mg/ml).

16. BCIP (dissolved in 100% dimethylformamide, 50 mg/ml).

17, 100 mmol / LTris-HCL (pH 9.5).

18. 100 mmol/L NaCl.

19, 50 mmol/LTris-HCL (pH 7.5), 5 mmol/L EDTA.
(See molecular clones)

Fourth, the main steps on the Internet can be collected almost all Western Blot Chinese and English data, for experimental reference only, can be adjusted according to the actual situation of their own experiments.

V. Common problem guides for experiments are mainly divided into the following categories according to the types of problems (the following data rights are for reference, please do not blindly imitate!):

1. Reference book recommendation

A. What information is better for beginners?
Answer: The "Experimental Guide to Antibody Technology" and Antibodies (a laboratory manual, written by Ed Harlow, david lane) are good.

2. Frequently asked questions about samples

B. Western Blot (hereinafter abbreviated as Western Blot) for mitochondrial membrane UCP2 protein, frozen after extraction of mitochondria (without protease inhibitor), the first antibody of Dr. Deer used, and there are still traces, which are getting worse and worse. The amount of sample has been added to 120μg, and it is still not possible to change the primary antibody of santa cloz. what is the reason? Is the protease inhibitor single plus PMSF?
Answer: Suspected to be a sample problem, it may be: 1, the sample can not be frozen and thawed repeatedly; 2, the sample is not added with protease inhibitors. At the same time, it is recommended to check the Western Blot process to increase the concentration of the primary antibody. For the addition of protease inhibitors, it is generally possible to add PMSF, and it is preferable to add a few protease inhibitors.

C. Cellular level to do Western Blot, how many cells are enough for Western Blot?
Answer: Generally 5*106 is enough.

D. Can the same sample simultaneously extract RNA and protein, does this have any effect on Western Blot?
Answer: Yes, no problem, we have done it.

E. Can the same protein sample be tested by Western Blot of both factors simultaneously?
Answer: Of course, some can even measure dozens of samples at the same time.

F. If the target protein is a membrane protein or a cytoplasmic protein, what should you pay attention to when operating?
Answer: If it is a membrane protein and a cytoplasmic protein, the detergent used should be much milder. At this time, it is best to add NaF to inhibit the activity of the phosphorylase.

G. My sample has a very low protein content of less than 1 microgram per microliter, but it is often found that only a part of the protein is transferred to the membrane during the film transfer, that is, all the protein strips found in the gel after the film is transferred. The belt is all there, but the color is faded. Is there any way to solve it?
Answer: You can increase the amount of sample loading, no problem, and you can reduce the current to extend the time when transferring, add 5-10% methanol.

H. The protein to be separated is 260kd in molecular weight. What is the appropriate concentration of separation gel for SDS-PAGE electrophoresis? What is the concentration of the laminated glue? Is such a large molecular weight protein easy to use as a Western Blot?
Answer: 260kd protein is not good, 6% separation gel, Stacking Gel 3.5%.

I. If the sample load is overloaded, what method should I use to increase the sample load? If you need to increase the amount of sample, the original weak band can be seen clearly.
Answer: You can concentrate the sample or dialy off a small amount of small protein based on your 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 comb.

J. How long can the protein be stored after denaturation?
Answer: -80 ° C, no problem for one or two years. The two most important two: don't be hydrolyzed by proteases; don't be digested by bacteria (also hydrolyzed by enzymes).

K. The molecular weight of the protein I measured is 105KD. It is reasonable to say that the separation gel should be 7.5%. However, the data I have checked require that the separation gel and the concentrated gel all adopt 11% formula. I don't know why?
Answer: The two gels mentioned above can be used because the 105KD protein is within the linear resolution range of the above two gels, but pay attention to the strip position.

L. Next I am going to use DAB color development technology, the secondary antibody is a biotinylated polyclonal antibody, and the third antibody is avidin biotin system. I don’t know if the blocking solution needs to be adjusted after using such a solution. With 5% skim milk powder? It seems that there is information that skimmed milk powder will affect the production of avidin biotin, is it?
Answer: Skimmed milk powder cannot be used because it contains biotin in skimmed milk powder. It should be better to replace it with BSA.

M. There is still a question. What is the total amount of protein that is normally loaded at a time, is it related to the expression level of the target protein?
Answer: Western Blot generally loads between 30 and 100 micrograms. The results are related to the abundance of the target protein, the amount of sample loaded, the amount of secondary and secondary antibodies, and the time of incubation. It is also related to the length of coloration. When you start to touch the condition, in order to get a positive result, each step can be a little longer and longer, of course, the background will come out. To get good results, if the antibodies are good, it is easier. If the antibodies are not good, you need to try them repeatedly. Of course, some of them are not suitable for Western Blot. So getting good results is not easy.

N. When doing the western sample of the tissue sample, what is the flaw in the sample? Also, have you used large bovine serum as a sealer? What is the concentration? Is the effect better than BSA?
Answer: Grinding, homogenization, and sonication are required. The solubility of the protein is better, the centrifugation is sufficient, the membrane protein needs to be extracted by a more violent method, and the low-abundance membrane protein may be extracted step by step (ultracentrifugation). Another point is that the protease activity in the tissue is stronger, and it is necessary to pay attention to inhibiting the activity of the protease (adding PMSF and protease inhibitor cocktail), and the blocking agent is generally used as 5% skim milk powder. If the primary antibody is a polyclonal antibody, BSA is also a good choice.

O. Can you introduce the large molecular weight protein 200KD, what should you pay attention to when doing western?
Answer: When doing Western Blot of 200kd protein, it is necessary to choose >7% of the separation glue; be careful when stripping the glue; the transfer time needs to be extended accordingly; do the molecular weight reference (otherwise, the miscellaneous band does not know how to analyze).

P. Is there any way to increase the amount of sample?
Answer: The sample can be concentrated; increase the sample volume to increase the sample load.

Q. The target protein I want to detect is a membrane protein with a molecular weight of about 42kd. Is it possible to extract membrane proteins without using an ultracentrifuge? Is there a method for referring to membrane proteins directly with a low-temperature high-speed centrifuge? The protein molecular weight of 42kd Is it big?
Answer: If you need to extract membrane proteins (rather than just extracting membrane proteins), you can use Ripa buffer to extract membrane proteins and cytoplasmic proteins. Use this as a Western Blot. If it is necessary to extract only membrane proteins, an ultracentrifuge is used. 42kd is not too big, it is not too small, so it can be implemented according to the general transfer method.

Is there any specific requirement for the amount of R. protein loaded?
Answer: The sample load should be determined according to the requirements of the experiment. If the requirement is quantitative and semi-quantitative Western Blot, the sample load should be equal. If it is only qualitative, there is not much relationship. Try to do as much as possible, but not More than 0.3μg/mm2.

Is the ratio of S. primary antibody and secondary antibody important?
Answer: It is more important to adjust the ratio of primary antibody to secondary antibody. You can remove some non-specific background.

3. Antibody

T. Do cell signaling, to do a phosphorylation factor Western Blot, what are the requirements of the secondary antibody?
Answer: There is no requirement for secondary antibody. It depends on your experimental conditions to choose. It is generally recommended to use HRP-labeled secondary antibody.

U. Another antibody from the same company is very effective with this dilution, so I didn't do a pre-test. I was afraid of time. What kind of dilution is better? I used the ELL+plus kit to develop color. The membrane was incubated overnight, and the primary antibody was also incubated overnight. If it was closed overnight, it would take four days to see the results.
Answer: Different antibodies, even the same company's antibodies, the best antibody dilution is not the same, you need to experiment. I don't think it is necessary to transfer the film overnight. The purpose of the film transfer is to transfer the protein to the film. Why waste time? As for the specific film transfer time, it depends on the molecular weight of your target protein; the equipment for film transfer is semi-dry or wet. The primary antibody can of course stay overnight. If you want a short Western Blot time, you can increase the incubation temperature of the primary antibody. Our laboratory is usually 37 degrees, two hours is enough; you can refer to the antibody instructions. As for the dilution of the primary and secondary antibodies, you can try 1:500 for the primary antibody and 1:200 for the secondary antibody. In addition, it is recommended that you wash the film several times, preferably several times. It is best to be closed; after the primary antibody and the secondary antibody are at least 5x5min, it is not easy to run a good film. Do not waste your time, this will not waste your time, only Will save you time!

Can V. immunohistochemistry and Western Blot use the same antibody?
Answer: When 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 Both proteins and boiled proteins are contained; conformational epitopes are lost due to the spatial structure of the protein and post-cooking denaturation. If the antibody you are using recognizes several amino acids in a protein, that is, a linear epitope, then the antibody can be used for both immunohistochemistry and Westernization, and if the antibody recognizes a conformational epitope, it can only be used. Immunohistochemistry. The amino acid range indicating the recognition of such antibodies is indicated in the general antibody specification. (limited to monoclonal antibody)

Repetitive application of antibodies in W.Western Blot: 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.

4. Problems with filter paper, glue and film

X.NC film \ PVDF film \ How to identify nylon film?
Answer: Nylon membrane is an ideal nucleic acid solid phase support. There are many types; nitrocellulose membrane is the most widely used solid phase support at present, and the price is the cheapest; PVDF membrane is somewhere in between.
In terms of binding ability: nylon membrane can bind DNA and RNA up to 480-600μg/cm2, and can bind nucleic acid fragments as short as 10bp; nitrocellulose membrane can bind DNA and RNA up to 80-100μg/cm2, for 200bp The binding ability of nucleic acid fragments is not strong; the ability of PVDF membrane to bind DNA and RNA can reach 125-300μg/cm2.
In terms of temperature adaptability: after the nylon membrane is baked or irradiated with ultraviolet light, a part of the pyrimidine base in the nucleic acid can bind to the positive charge on the membrane; the nitrocellulose membrane binds to the DNA by hydrophobic interaction, and the binding is not strong; PVDF The membrane is firmly bonded and resistant to high temperatures, and is particularly suitable for Western blotting.
In terms of toughness: the nylon membrane is strong; the nitrocellulose membrane is brittle and easily broken; the PVDF membrane is stronger.
In terms of repeatability: the nylon membrane can be repeatedly used for molecular hybridization. After hybridization, the probe molecules can be eluted by alkali denaturation; the nitrocellulose membrane cannot be reused; the PVDF membrane can be reused.

Y. When doing Western Blot, the purpose of PBDF membrane soaking with methanol?
Answer: The purpose of the PVDF membrane with methanol bubbles is to activate the positively charged groups on the PVDF membrane, making it easier to bind to negatively charged proteins. This is also the purpose of adding methanol to small molecules.

Z. Can I detect phosphorylated JNK and non-phosphorylated JNK on the same membrane?
Answer: Yes.

AA. After the film is dyed by Ponceau red, why does the transfer of one end of the large protein molecule (that is, the side of the spotted space) does not seem to be very good, why?
Answer: This is normal. The protein transfer of macromolecules is slow. When you extend the transfer time and current, the macromolecule will be much better at one end, but the small molecule may become lighter.

BB. I would like to ask you to use the three decontamination lysis method for lysing cells, or use the loading buffer?
Answer: Using a loading buffer, there are several advantages to extract total protein while inactivating phosphorylase.

CC. What is the use of tank system?
Answer: It is recommended to low voltage, long time, (usually the tank system uses a good pressure), such as 28V 14-16hrs.

DD. Do HSP WESTEN quantification, the same antibody immunohistochemistry can be made, but WESTEN can not?
Answer: This is mostly a question of antibodies. It depends on the description of the antibodies. Can you do Western Blot and IHC?

How should EE. membranes be treated?
Answer: Generally, you can use methanol bubbles.

FF. If it is a 6×8 transfer film, how many primary antibodies should I add?
Answer: The dilution of the primary antibody is explained. You can see it according to your primary antibody, but the large membrane incubation volume is usually at least 3-5ml.

GG. What are the requirements for the upper and lower tank buffers? How can we achieve the best results?
Answer: No requirements.

HH. What is the reason why the glue used in running electrophoresis is always "shrink"? Is there something wrong with it?
Answer: No problem, the moisture in your glue is evaporated. Wrap the plastic wrap over the night and add some water to keep the humidity inside. If it is overnight, the moisture in the gel is evaporated, and it can be wrapped in plastic wrap. It may also be a problem with the mother liquor (30% polyacrylamide). You can re-form an observation; replace the reagents, try to change them, choose good. Reagents to avoid trouble finding problems. Too high a level of methanol in the decolorizing solution can also cause gelation.

II. What is the size of the film, filter paper and glue?
Answer: If you are using a semi-dry run, the order is: Cathode - "Filter Paper -" Glue - "Film -" filter paper. The length and width of the filter paper are 1-2 mm smaller than the glue, and the length and width of the film are 1-2 mm larger than the glue. Absolute taboo: The upper and lower layers of filter paper are in contact with each other because they are too large, which will short circuit and the current will not pass through the glue and filter paper.

The molecular weight span of JJ. protein is very large. If you want to separate small 21KD, medium to 66KD, and up to 170KD, can you do it at one time?
Answer: Such a wide distribution is not easy to transfer. Generally recommended: 21kd and 66kd can be transferred together, 12% SDS-PAge, wet to 36V, 3-5hrs, can be adjusted according to your laboratory experience; 170kd with 7% SDS-page, 48V 10hrs-16hrs.

KK. It is not good to transfer large molecular weight proteins to the membrane. How to solve the problem of low transfer efficiency?
Answer: Consider: adding 20% ​​methanol to the transfer buffer (refers to the final concentration) (optimized transfer buffer, refer to the Protein Technology Manual), because methanol can reduce protein elution efficiency, but can increase protein and NC Membrane binding ability, methanol can prevent gel deformation, methanol can prolong the transfer time for high molecular weight protein; transfer buffer is added to the final concentration of 0.1% SDS, also to increase transfer efficiency; use high quality transfer film, or use small pore size NC Membrane (0.2 micron); cross-linked with glutaraldehyde; low-concentration gel, as low as 6%. If too large, you can also consider using agarose gel; increase the transfer voltage / current; increase the transfer time.

LL. How to choose the most suitable protein hybrid membrane?
Answer: Western blot hybridization is a very common technique used in molecular biology experiments. Choosing a quality hybrid, suitable, and convenient hybrid membrane is an important part of the success of this experiment. According to the hybridization scheme, the characteristics of the transferred biomacromolecules, and the molecular size, we must tailor the material, make a reasonable choice from the material, pore size and specifications of the hybrid membrane.
Nitrocellulose membrane: The nitrocellulose membrane is the standard solid phase support for Western blot experiments. In the low ion transfer buffer environment, most of the negatively charged proteins will combine with the nitrocellulose membrane to form a high affinity, although the mechanism is not very clear, but due to the nitrocellulose membrane This feature, and easy to block non-specific binding, has been widely used. The bound protein can also be eluted under the action of a non-ionic detergent. Depending on the molecular weight of the protein to be transferred, a nitrocellulose membrane of different pore sizes is selected. Because as the membrane pore size continues to decrease, the membrane binds to low molecular weight proteins more firmly. However, if the membrane pore size is less than 0.1 mm, protein transfer is difficult. Therefore, we usually use nitrocellulose membranes of both 0.45 μm and 0.2 μm sizes. A protein larger than 20 kD can be used with a 0.45 μm membrane, and a protein smaller than 20 kD is a 0.2 μm membrane. If a membrane of 0.45 μm is used, a "Blowthroμgh" phenomenon occurs. From the texture of the membrane, the most important indicator is the amount of protein that can be bound per unit area. The binding capacity of the nitrocellulose membrane is mainly related to the purity of the nitrocellulose of the membrane. Some nitrocellulose membranes on the market usually have a large amount of cellulose acetate, thereby reducing the amount of protein binding. S&S uses 100% pure nitrocellulose to ensure maximum protein binding, up to 80-150μg/cm2. Due to the 100% purity, non-specific binding is also greatly reduced, the hybridization background is reduced, and a high stringency elution step is not required. Second, the strength and toughness of the film are also factors to consider. Conventional nitrocellulose membranes are relatively brittle, and they are broken once or twice, and cannot be used repeatedly.
PVDF transfer film: PVDF is a high-strength, corrosion-resistant material that is commonly used to make water pipes. PVDF membranes can bind proteins and can separate small fragments of proteins, initially used for protein sequencing, because nitrocellulose membranes degrade in Edman reagents, so PDVF is sought as a substitute, although PDVF membrane binding The efficiency of the protein is not as high as that of the nitrocellulose membrane, but because of its stability and corrosion resistance, it has become an ideal product for protein sequencing and has been used ever since. Like the nitrocellulose membrane, the PVDF membrane can perform various dyeing and chemiluminescence detections, and has a wide range of applications. This PVDF membrane has higher sensitivity, resolution and protein affinity than conventional membranes in a fine process, making it ideal for the detection of low molecular weight proteins. However, the PVDF membrane must be soaked in pure methanol for 1-5 seconds before use.
Ion exchange type transfer membrane: nitrocellulose and PVDF membranes bind to proteins by hydrophobic interaction, and a type of membrane binds to biomacromolecules according to ion exchange. A DEAE anion exchange membrane made of DEAE (diethylaminoethyl) modified cellulose can also be used as a solid support for Western blotting. DEAE can effectively bind anionic groups, including those above its isoelectric point. Below pH 10, the DEAE group is chargeable and binds to protein molecules in low ionic strength transfer fluids. Its optimum pH environment is 5-7. DEAE membranes can be used for the study of proteoglycans, viruses, enzymes, and hemoglobin. This 0.45 μm pore size DEAE membrane can be used for nucleic acid binding studies in addition to Western Blotting.
Another ion exchange membrane is a carboxymethyl (CM) modified cellulose membrane that binds to protein and polypeptide molecules, as well as other positively charged samples, with a pH range of 4-7. The bound polypeptide molecules can be eluted from the CM membrane for amino acid series analysis or microsequencing.

5.Marker related questions

MM. I use the visual marker (BIO_RAD), but the electrophoresis does not run all the 8 bands. What is the reason? How to improve? Glue used 8%, 10%, 12%, this is the case. The marker is new.
Answer: In general, the small molecular weight Marker runs away, increasing the gel concentration or reducing the electrophoresis time. Of course gradient glue is also a good choice.

NN. uses a marker consisting of 100kd, 75kd, 45kd, 30kd, 20kd, 10kd from Roche molecular Biochemicals. Marker can also be seen when starting Western Blot, and of course only three of them can be seen. SDS-PAGE electrophoresis was carried out at 80 V, and transferred with a constant pressure of 10 V for 45 min. I did not perform Ponceau staining when I did Western Blot a few times, but even with this method I can only see that the marker has a strip of about 30KD. Then, the two target proteins of 70KD and 130KD were simultaneously analyzed on a piece of gel, and analyzed by the medium sample. Instead of using the indirect method, the enzyme-linked antibody of the V5 epitope of the C-terminus of the fusion protein was directly used (Anti-V5- HRP). But it is not the result, I am very upset. Thank you for giving me some advice!
Answer: 1. “I used the marker of 100kd, 75kd, 45kd, 30kd, 20kd, 10kd from Roche molecular Biochemicals. I can see the marker when I start Western Blot. Of course, I can only see up to three of them. "Sometimes, the Prestained Marker will be worse after a long time, and the electrophoresis is unclear and diffuse. But your problem may have other problems, it may be that the protein is not tightly bound to the membrane. The transfer is to add more methanol.
2. “There was no Li Chunhong staining when I did Western Blot, but even with this method, I can only see that the marker has a strip of about 30KD.” The semi-dry method is recommended to use constant current during transfer. This is also a good transfer of 30kd-50kd.
3. "There is another analysis of the two target proteins of 70KD and 130KD on a single gel. The medium sample is used for analysis. Instead of the indirect method, the enzyme-linked antibody of the V5 epitope of the C-terminus of the fusion protein is directly used. -V5-HRP)."
Whether the expression level is detected, whether the secondary antibody is good, do you have a positive control? The best transfer time for you to make such a large protein is extended to 1.5hrs.

6. Selection of dyeing

What kind of dyeing is OO.Western Blot?
Answer: (1) Anionic dyes are the most commonly used, especially amino black, which is fast decolorizing. The background detection limit can reach 1.5μg. Although the horse has the same sensitivity as amino black, it has slow decolorization and high background. Ponceau S and Fast Green are easily removed from the protein after detection for subsequent amino acid analysis. The disadvantage is that methanol in the solvent system can cause shrinkage or destruction of the nitrocellulose membrane. I can't use a film that is congratulatory. Low sensitivity.
(2) Colloidal gold, high sensitivity, detection range can reach pg level, but the dyeing ratio is stable.
(3) The biotinylation sensitivity is between 1, 2 and can be used for any kind of membrane.

7. Reference question

PP. Is the Western Blot experiment semi-quantitative must add ACTIN internal reference?
Answer: For the experiment of publishing an article, it is best to add internal reference and the experiment is rigorous.

QQ. Is the result of analysis using BANDSCAN?
Answer: Analysis of the general results is no problem.

RR. What is appropriate for the internal Blot internal antigen selection?
Answer: Histone can be used. The expression of histone in the nucleus is very stable. Many of them can be used as internal reference. You can select the internal reference you want on the Internet.

SS. Whether the current is more accurate than the voltage when the film is transferred. Is it based on 0.8 mA/cm2, usually about 1 hour?
Answer: No, the semi-dry method recommends constant current, and the current and time are generally determined according to the size of the protein of interest.

TT. Do a semi-quantitative Western Blot of human ovarian cancer cell lines, internal reference B-actin, GAPDH, which is good?
Answer: You can use beta-actin.

8. Frequently Asked Questions for Buffer Formulations

UU. What is the antifoaming agent A used when the skim milk powder after film transfer is closed? Is Tris-HCl just blended with Tris and hydrochloric acid?
Answer: The skimmed milk powder blocking solution after transfer is 5% TBST skim milk powder. Tris-HCl is a configuration in which the Tris salt is adjusted to pH with HCl.

VV. Prepare the Western Blot of the rat brain. The protein is located in the nucleus. What is the extract and operation method of this protein? Must every step be low? This protein is a phosphorylated protein, how to prevent dephosphorylation during operation?
Answer: You can use the buffer to extract the total protein to raise the nuclear protein, you can add NaF to prevent dephosphorylation.

WW. Would you like to ask if there are any principles for cell lysate selection of protease inhibitors? Not affected by the source of the organization? Is there a difference between the cell membrane and the cytoplasm?
Answer: In general, it is sufficient to add a spectral protease inhibitor when extracting, and keep it low during operation. Unless there is a specific specification in the literature, there is generally no difference.

XX. Recently, I made two Western Blots. Not only did I have no positive results, but there was no coloration background. Both electrophoresis and transfection were stained and stripped. The film and the color developing solution and the DAB coloring solution were all tested, no problem. 1. Detect GAD--molecular weight 67kd, extract sucrose, and the same three decontamination lysates. Does sucrose have no effect on it? Samples - -20 degrees placed within one week of testing. 2, the primary antibody placed for 2 years, may not be high titer! It is 1:100. If it is the reason for the primary antibody, will not be the background? 3, the first time there is uneven background, because the primary bag is not uniform when the overnight stay. No background color after the second time. 4. The blocking solution is made of 15% skim milk powder TBST, the rinsing liquid is TBST solution containing 1% BSA, and TWEEN-20 is 0.1%. It is not a problem of blocking liquid.
Answer: You can find the reason for the following questions. 1. Block the solution with 5% Milk, rinse solution (washing buffer with TBST) 2. See if the primary antibody works, drop to 1:20. 3. See if there is a problem with the secondary antibody.

YY. What is the purpose of adding methanol?
Answer: Adding methanol has a certain fixed effect, because small molecules of protein are easy to transfer out (especially on nitrocellulose membranes, because NC membranes have weaker ability to bind proteins).

ZZ. “The skim milk powder blocking solution after transfer is 5% TBST skim milk powder”, and the last T of TBST is Tween, what is the concentration?
Answer: Tween, the formula is as follows: Tris-Buffered Saline Tween-20 (TBST), Dissolve 8.8g of NaCl, 0.2g of KCl, and 3g of Tris base in 800ml of distilled H2O, Add 500ul of Tween-20, Adjust the pH to 7.4 with HCl, Add distilled H2O to 1L, Sterilize by autoclaving.

AAA. Is there any regulation for the temperature of the closure, primary antibody, and secondary antibody? For example, do I do it at room temperature now, or at 4 degrees?
Answer: Both can be performed at room temperature. If the time is not enough, the primary antibody can be incubated at room temperature for one hour and then at 4 degrees overnight.

BBB. The laboratory has no NP40. Can I use sds? Has there been any extraction of membrane proteins with urea and thiourea? The formula is as follows: 7M urea, 2M thiourea, Triton-x-100 0.2ml, fresh addition: 65mM DTT
Protease Inhibitor: Kit HaltTM Protease inhibitor cocktail kit 1%(v/v)
It is a formulation for extracting proteins for two-dimensional electrophoresis. Is it not only feasible to extract whole protein from cells (mainly want to get the membrane protein)?
Modified RIPA lysis buffer (Tris.HCl, 50 mmol/L, pH 7.5; NaCl, 150 mmol/L; NP-40, 1%; sodium deoxycholate, 0.5%; SDS, 0.1%; EDTA, 1 mmol /L; PMSF, 1 mmol/L; Leupeptin, 2 μg/ml) What is the effect on membrane proteins? In addition, is EDTA in this side used as a protease inhibitor?
Answer: NP-40 is definitely not recommended for 2-D (because even if the imported NP-40 is not pure, the impurities will affect the mass spectrometry results). For animal cells, the protease activity is weak (relative to tissues and E. coli, etc.), and cocktails may not be used, because the denaturing environment composed of 7M urea + 2M thiourea + 4% CHAPS is sufficient to inhibit most protease activities, 2M thiourea + 4 %CHAPS is very helpful for extracting membrane proteins, but if you are working full-time on membrane proteins, it is recommended to use fractional extraction. Gradient centrifugation and some detergent based methods can also be used. EDTA is used to inactivate metalloproteinases (primarily for its incorporation). Adding too much protease inhibitor can lead to protein modification. It doesn't matter if you do WB. When you do MAILDI, it will bring trouble to the correct identification. Ampholyte is absent from the lysis buffer (in 2-D lysis buffer, the role of the ampholyte: providing a continuous pH gradient that greatly increases the solubility of the protein; it also removes a portion of the nucleic acid); SDS is recommended because SDS will combine with protein to cause its isoelectric point to change. If you really want to use it, the final concentration will drop below 0.1%.

CCC.Western Stripping Buffer formula solution: METHOD1
1, stripping buffer: 62.5mmol / l Tris PH6.7; 100mmol / l beta-mercaptoethanol; 2% SDS
Secondary illumination protocol: 1.stripping buffer wash film: 50-degree water bath for 30 minutes, shake on a shaker for 10-20 minutes.
2, 1 * PBST wash: shake the shaker for 30 minutes.
3, closed, plus primary antibody, secondary antibody (same as the first light)

METHOD2
(50 ml total): ?-mercaptoethanol 342 μl; 20% SDS 5 ml; Tris-Cl pH 6.7 3.125 ml; add ddH2O to 50 ml.
Method: The used membrane was immersed in a stripping buffer, placed in a 50 ° C water bath for 30 min, and shaken intermittently. Then wash it with TTBS for 3*5min. At this point you can use the new transferred film again.
The advantages of this method: save trouble, save effort, save money, in line with international practice.

METHOD3
1, beta-metaptoethanol 35ul
2, 10% SDS 1ml
3, tris (0.5M, pH6.7) 625ul
4, dH2O 3.34ml
50-55 ° C, 30 min.

METHOD4
The stripping buffer should be able to be placed for a long time. However, I am used to the match - after all, adding beta-mercaptoethanol is too bad after use, and it is used; and, with the ready-made Tris-HCl buffer and SDS mother liquor, it is very convenient. Every time I use 5ml less, I use 50ml each time.

METHOD5
0.5 M NaCl, 0.5 M HAc; shaken at room temperature for 15 min.

METHOD6
将用过的膜泡在1*TBS中室温振摇过夜，中间可以换液2-3次，然后封闭，加一抗，二抗（同第一次发光），实践证明方法完全可行。

不管用那种方法，洗脱后都要用PBS或TBS再洗几次。

9.条件的摸索

DDD.用的是Santa Cruz的抗体，也实验过一抗和二抗肯定能结合，二抗加DAB肯定能显色。电泳的胶用考马斯亮蓝染色没问题，但是不知道与Marker对应的条带是否是我要的（我目的蛋白的分子量分别是55KD、29KD）。半干法2小时转膜后，丽春红染色发现大分子量蛋白转过去的较少。难道是裂解液出了问题？我用的是三去污剂，但没加叠氮钠和大概叫Apoptin的那种蛋白酶抑制剂。冰上裂解-80度冻存的细胞，4度12000g离心5分钟，取上清，与分子克隆（第二版）上的加样buffer混合，沸水变性5分钟，上样。不知道是哪里出了问题？
解答：建议：1、首先确定您提的蛋白质量如何？可用PIERCE公司的BCA试剂盒测蛋白的浓度，一般来讲，其浓度应该在几-20微克/微升。
2、若是蛋白没问题，哪就看是不是电泳的问题，首先要看胶的浓度，您目的蛋白的分子量分别是55KD、29KD，建议分别用10％和12％的胶。60-80V，1小时左右。跑过积层胶与分离胶的线时，换用100V，3-4小时。
3、转膜，建议恒压，15V，不用转2小时，45分钟足以。您所说的大蛋白转过去的，并不是真正的少，而是因为在提的蛋白中大蛋白本身就很少。我曾经也转过2小时，但和45分钟的区别并不大。
4、根据MARKER的条带（我的是7条带：14、18、25、35、45、66、116KD），您根据MARKER的条带剪下25与35之间（29KD）的条带，45-66之间（50KD）的条带。这样第一，可以节省抗体，第二，您要的目的条带肯定在上面。
5、延长1抗、2抗孵育时间（我曾室温1小时，4度过夜），适当加大1抗浓度。
6、我买的也是Santa Cruz的抗体，我觉得质量还可以，我想您应该先找其他方面的原因。

EEE.电泳用的是恒流，一块胶，20mA，100分钟左右。转膜也是恒流，38mA，100分钟。而且我用别人的细胞和一抗在我的整个反应体系下做出来了，当然彼此的目的蛋白不同。所以，我想问题应该出在抗原和一抗上，不知对不对。
解答：电泳的条件：样品的分子量决定了胶的浓度，一般使样品跑至胶的中部即可。正常条件下，电泳时溴酚蓝和10kd左右蛋白跑在一起。由此可以决定电泳的电压和时间。建议你用恒压80－100伏。

FFF.BIO-RAD的半干转运系统有一个很致命的弱点就是无法控温（我用的就是这种），当电流过高，而系统的散热又比较差，滤纸的吸水性比较差的情况下，就很容易烧胶。
就转膜时，是采取恒压还是恒流的问题，我想和大家探讨一下，我感觉我这个系统用恒流很容易烧胶，我的胶有68cm2，用50mA恒流来转膜，刚开始电压就很高，有20 v左右，而用恒压，开始电流有110mA，但15min后，电流就降到8OmA，30min后就稳定在40mA，不就相当于恒流吗？
解答：恒流时电压逐渐升高的原因是湿滤纸逐渐变干因而电阻逐渐增大的缘故，如果电压升得太快，可以使滤纸更湿一些以克服。就我的感觉，20V的电流30min以后20Kd以下的分子丢失很多，不过我用的是小胶40cm2，不知有无不同。

GGG.我想尽量提高转膜的效率（我的实验要求转到膜上的蛋白越多越好，不管是什么大小蛋白）不知道有那些办法？
解答：不管怎么转都会存在蛋白转移不完全（电压过小时间过短）或过度转移（电压过大时间过长）的问题，鱼（小分子量蛋白）和熊掌（大分子量蛋白）不可得兼呵呵。建议把胶切成两半，比如以35KD为界，分别进行转膜，下半时间短，上半时间长一点，应该会好一些。

HHH.请问一下PVDF膜和硝酸膜结合蛋白的原理是什么？
解答：一般而言，硝酸纤维素膜是通过疏水作用来和蛋白质相联，这样的话，反复洗几次后，蛋白容易掉下来，结果较差。尼龙膜主要通过它膜上的正电荷和蛋白接合（注：常用的PVDF即带正电荷的尼龙膜），同时也有疏水作用，但相对较弱。这样的话，PVDF膜和蛋白接合较牢，不易脱落，结果较好。

III.1、煮好后的样品，若没有及时上样分离，应如何保存，可以保存多久？2、有没有人在用bio-rad的小型垂直电泳槽，有没有操作手册？3、湿式转移时是否必须要用bio-rad的专用滤纸？4、恒压转移的条件如何确定，因为我要分离小至21KD，中至66KD，大致170KD的蛋白质，转移条件能够相同吗？5、凝胶的浓度是不是可以用一个浓度？书上写不同的凝胶浓度分离的分子量范围不同，还给出了一个线形范围，是不是不在这个范围内也能分离，只是就不是线性范围了？
解答：1、煮好后的样品，放到-20，我们在一个月后此样品，效果一样。
2、bio-rad的小型垂直电泳槽的操作手册在他们的主页Bio-Rad USA上有。
3、转移时一般的WATERMEN滤纸就可以。
4、转移条件是和蛋白质大小有关的：以次确定电压和时间。具体可让ptglab帮你定夺。
5、凝胶的浓度也是和分离蛋白质大小有关。不是随心所欲选的，否则分离效果可能不是你所期望的。

JJJ.怎样设计实验来确定最佳的条件？
解答：随便说一点， 具体的还是需要自己想：
1、在每个上样孔里上同样的蛋白样品，量也一样，最好是组织样，（也可以跑1 个大well， 不插梳子，多上样，）SDS-page；
2、转移， 设定电流或电压；
3、每隔1（or n） 小时，取一点膜染色，看转移效果。

KKK.我要测两种抗体，一种为磷酸化的目的蛋白，一种为总的目的蛋白，不知道用什么方法strip最好，我用甘氨酸（PH2.9）漂洗15分钟，似乎没什么效果？
解答：你可以加巯基乙醇（loading buffer 一样的浓度），56度， 30mins，看看。

LLL.1、在用PBS洗涤抗原-抗体-ProteinA-Agarose复合物时，每次要重悬多长时间合适？2、最后用2xSDS重悬抗原-抗体复合物离心后，由于2XSDS中已经加入了溴芬兰，因此下面的Agarose珠子几乎看不到，所以吸取上清加样时也不知道里面是否吸进了Agarose。不知有什么方法可以解决这个问题，或者即使吸进了一些Agarose也不要紧呢？
解答：1、不用重悬多久，重悬起来了就可以离心了。
2、加2X BUFFER前大体上已经知道有多少胶粒了，吸到那个位置时小心点就是了。我也试过一些次，首先离心稍微长一点，长20秒吧，希望胶粒能沉得结实点（我想象的），再吸取。如果感觉枪头不是很顺畅的时候可能就是碰到胶粒了。很难一点胶粒也吸取不上来的，尽力做好就是了。

MMM.磷酸化抗体的检测样本制备时是否一定要加NaF等？
解答：NaF是一种广谱磷酸化酶的抑制剂，一般最好加。但是不加也可以，大部分时候是不用加的。我做的时候从未加过，都做出来了。

NNN.Western Blot中block的最短时间?
解答：每一步1小时足够了， 中间换抗体要洗的话多换液几次，每次时间10分钟就够，洗3次只要半小时。跑胶1小时， 转移1小时，block半小时就行，1抗1小时，洗半小时，2抗1小时，洗半小时，显色10分钟。一般跑两块胶，一块染色， 一块western。一天肯定完事，一般不用等到第二天。

OOO.想用Western检测基因转染后细胞培养上清中表达的目的蛋白（定性），分子量为20KD，浓度约为几百ng/ml。蛋白样品需浓缩、纯化吗？如何浓缩、纯化上清液中的目的蛋白？对小分子蛋白Western blot时需特别注意哪些条件？
解答：按照你提供的浓度，如果做Western Blot，是不用浓缩样品的. 对于20kd的小分子的蛋白，Western Blot中要注意的是：
1、转移时的时间，
2、转移时的电流或电压.
3、transfer buffer 中加20%的甲醇.
4、可以用13-15%的分离胶.

PPP.蛋白分子量大小分别为21kd、28kd，用的是湿转，请问多大电流，多长时间比较合适？
解答：分子量比较小，最好是用干转，湿转效率太高，易转过了。干转的话，用2.5 A/cm2， 30min就应该够了。湿转，按照bio-rad的说明，用100mA，也得要半个多小时吧。

QQQ.需要测同一种蛋白质的总量与磷酸化的量，但相互间干扰太大，怎么办？
解答：将膜放入stripping buffer（SDS 2%，Tris·Cl (PH=6.7) 62.5mM，beta-巯基乙醇100mM）中，50℃孵育30分钟，TTBS西三次，再重新加入一抗，进行另一种抗原的检测。

RRR.做Western Blot实验时，发现转膜时的电流总是偏小，转膜的效率也偏低. 100V恒压转膜时的电流只有190mA左右，而以前都有250mA。体系和条件都和以前一样，只是环境温度比以前低了很多。
解答：有可能重复使用了转移缓冲液，随着离子的逐渐减少，电阻越来越大，当然恒压时电流越来越小了。建议更换转移缓冲液。反复使用不要超过三次。环境温度低是有利于转移的。

SSS.我电泳用的是恒流，一块胶，20mA，100分钟左右。转膜也是恒流，38mA，100分钟。而且我用别人的细胞和一抗在我的整个反应体系下做出来了，当然彼此的目的蛋白不同。所以，我想问题应该出在抗原和一抗上，不知对不对。一抗我买的是单抗，推荐的稀释度是1：100——1：1000。我用的是1：100。难道非要用多抗吗？按道理单抗也应该出条带呀！细胞用三去污剂裂解的，没有做定量，加巯基乙醇变性后，每孔上样20微升。提出的蛋白我保存在4度了，这样行吗？
解答：1、建议您用恒压进行电泳，先用70V，等跑过积层胶与分离胶的界限时，换用90-100V，再跑3小时左右。2、转膜可用恒流，但要根据你的膜的面积及所要检测的蛋白的分子量的大小来确定电流的大小，一般来讲，0.8-3.0mA/CM2，分子量大的蛋白就用的电流大些，譬如，你膜的面积是30CM2，蛋白的分子量是80KD，那么你就可以以2.0mA/CM2的条件进行，你就可以60mA的电流进行。3、我觉得你的抗体应该没问题。4、你提出的蛋白怎么保存在4度呢？我们实验事都是保存在-20度的，提出蛋白分装保存在-20度。

TTT.目的蛋白是一种6KD的分泌性蛋白，RT－PCR就显示细胞中mRNA表达不高，估计将培养上清冻干浓缩后采用Western Blot还可能检测不出，但如果用western，是否一定要用0.2μm的膜？用Tris-tricine SDS－PAGE电泳，电泳时分别管制三层凝胶，分离胶用40％T丙稀酰胺（2.6％C）浓度为16.5％，另外两层都用30％丙稀酰胺，中间一层浓度为10％，积层胶浓度为4％，凝胶厚度1mm，转膜的条件试过30V70分钟，膜上可见到小分子蛋白marker的条带，似乎见到目的条带，上样量为60μg细胞胞浆总蛋白，转膜的条件怎么样合适？
解答：一定要用0.2μm的膜，并且转移的条件要摸索一下，小分子的Western不好做，要根据你的实验器材来定，一般你要是有prestained marker就可以参照一下，如果相应的分子量大小的marker转移的好就可以了。

UUU.怎样才能把胶跑的非常漂亮，泳道和band都能很直，是不是上样的量很重要，罐胶有什么技巧吗？想跑漂亮，是不是应该先小电压，再高电压，总体上电压小些会跑的好些?还有前面有人说电泳液平玻璃板会使电泳条带漂亮些?
解答：影响跑胶跑的质量，有以下几个因素：1、电压，小的电压会使胶的分子筛效应得到充分发挥。电压越小，条带越漂亮，浓缩胶80v，分离胶100v就能跑得很好。2、胶的均匀度，胶越均匀，条带越窄，分离越均匀。倒胶之前，一定要充分混匀，玻璃板一定要干净，双蒸水隔离时，一定要比较轻地加上去，避免稀释上层的分离胶，使胶不均匀。

VVV.为什么提高大分子量蛋白的转移的时候，小分子量蛋白会丢失一些哪?什么原因?
解答：小分子的蛋白在转移过程中，会透过膜去，所以大分子的上去以后，有一部分小分子的就透过去了。

WWW.上次转染了1.6*106细胞，收集，收集到了400微升体积，加6*loading buffer， 95度煮5分钟，-20度，交替3次，离心，上样，7%分离胶，先80v跑进分离胶，在100v电泳至溴酚兰出胶，biorad半干转PDVF膜，15v转30分钟，预染marker全部进膜，转移后的胶考马斯亮兰染，可见残留的蛋白带，大分子量多些.blot时，封闭用5%奶粉TTBS 1小时，抗体稀释液TTBS，一抗(1:10，单抗上清，2000年制备)1小时，二抗(1:2000)1小时，均在室温.结果，50kd的小分子显色，160kd大分子未显色。
原因分析及准备改进：转染大容量的质粒(融合蛋白的质粒)的效率会相对较低，大分子量表达也会相对较低，加上大分子量蛋白的转移不完全，可能是我没有拿到大分子量条带结果的原因。另外背景稍微有一些脏(背景整体均匀一致)，估计是二抗的浓度高了些，准备降至1:5000，另外抗体稀释液准备改用5%奶粉TTBS，封闭改为室温2小时。这个过程有没有问题？
解答：首先分析你的整个实验步骤。我发现了两个比较大的毛病：
1、一抗用TBST稀释。按照我的理解，一抗最好用5%的TBST脱脂牛奶稀释，和你的封闭液相一致，这样可以降低背景。
2、“biorad半干转PDVF膜，15v转30分钟”，你是这么做的吗？因为我大部分时间是做的恒流转移，用的是0.1mA/膜，100kd--200kd转2小时。到最后电压会升到20v左右。你用恒压法，我不是很肯定你转30分钟能将大分子（160kd）的抗原转上去。我建议你最好能将时间延长，如果是恒流，可按我的做法；如果是恒压，可摸索一下，适当延长时间。有时候你的marker也有可能欺骗你，因为marker的量比较大，是很容易转上去的，实际上目标蛋白的量远远少于marker量。
我对你的结果分析如下：
1、你的结果很好，估计离目标不远了，很快就可以成功。
2、 没有160kd的带是因为你的转移时间过短，适当延长转移时间（我怀疑这是主要的问题）。
3、你的一抗用1：10，2抗可以降到1：5000，背景会低一点。

10.方法的介绍

XXX.我想将hbx转到hepg2 细胞里通过检测hbx蛋白的水平来检验转染的效率不知道可不可行？
解答：Western Blot检测HBX蛋白的表达来检测转染效率不是一个好办法，建议还是：
1、最好是带有荧光标签的HBX转染来看转染效率，最可靠
2、其次，HBX和荧光载体共转染，相对说明问题
3、荧光载体单独转染，主要是看细胞好不好转了呵呵转染效率高低荧光显微镜下一目了然了。有的细胞荧光强，有的细胞荧光弱，有的没有。所以HBX表达强很可能是少数细胞荧光强的结果，不代表转染效率。

YYY.半干法转移与胶的面积和蛋白分子两大小好像都有关系，那么湿法转移是不是所有的转移条件都一样呢？一般的条件是怎样的？
解答：半干转的电流大小是按照面积来算的，时间是根据蛋白分子大小定的；而湿转的话电流是恒定的，时间也是根据分子量而定。

ZZZ.转膜时何为湿法，何为半干法？
解答：半干法和湿法转移是两种不同的转移装置下的转移系统，将膜，胶，滤纸整个浸泡在buffer的Tank里转移的，叫湿法；用滤纸吸buffer来做转移体系的叫半干法。

AAAA.为什么浓缩胶和分离胶的电压不一致？同样的电压可以吗？
解答：浓缩胶的目的使得loading 的样品能够在同一条水平线上进入分离胶，如果电压太大前端的蛋白容易在后面的蛋白赶上来前进入分离胶。而在分离是理论上（实际上也是）小电压长时间分离的效果会更好，但是在操作过程中没有必要等那么长的时间，所以就用大电压快点跑。

BBBB.如果目的蛋白比较小，21和38kd，转膜时（Biorad的湿转仪）时间是否能缩短些？100伏电压，甲醇的量是否也可以相应增加？

解答：如果是小蛋白可以用小电压28V－36V，4－6hours，（4度）。甲醇10％－20％就可以了。

11.结果分析

CCCC.条带有时清晰，有时很散，不知为何？
解答：可能原因：样品可能存在降解；转膜不及时造成扩散；转移缓冲液甲醇浓度（ＮＣ膜可加到２０％，ＰＶＤＦ加到１５％）。

DDDD.显色目的条带又细又浅，靶蛋白是27KD的bcl-xl和67KD 的c-myc，应该是高表达。每个涌道上50-70μg蛋白，开始用半干转移，后来用湿转14v，16h，用PVDF膜转移。胶转移后染色检测，发现小分子量的基本转移走了，可是为什么条带老是不粗不浓呢?
解答：可能跟你的一抗有关系，还有应该高表达，那实际做的阳性对照是否是高表达；还有跟抗原量相关，你多上点蛋白会好的多；跟显色条件相关。

EEEE.背景很花，当然条带也很淡，如果我在显影液中多洗一会儿，背景就很深，以致无法辨认，但有时条带又很明显，背底很淡。
解答：这跟你washing的时间和强度有关，很可能你在这方面没有掌握好。显影时间是有范围的，久了肯定不行。

FFFF.纯化过的目的带包括其上下都出现了色带，而且对照菌也出现了条带，会是什么原因?
解答：一抗有问题，效价太低，且未纯化；表达量太低；提蛋白时可能出了问题。

GGGG.做Western Blot的检测的时候，用DAB显色还能看出条带，但是换成ECL的时候什么样结果都没有。我用的NOVA公司的发光底物，胶片是普通的X片，定影液和显影液都是别人留下来的，不知道是什么原因？
解答：一般的说，Ecl比DAB更灵敏，但是DAB是HRP最敏感的底物，所以有几种可能：
ECL底物失活了，你可以检测一下；敏感度正好不到ECl底物的范围。DAB能显色可不能催化ECL底物；显影液没用了。

HHHH.怎样分析结果，需要什么软件?
解答：如果你做定量或办定量，至少要用到一个光密度扫描分析软件，如果不是，直接分析就可以了。

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