1. Advantages of western blot
Answer: Sensitive, up to ng level, theoretically up to pg level with Ecl color rendering method. Convenient and high specificity.
2. Why is there no target protein in my cell extract?
Answer:
a) Do not express this protein in your cells, change to another cell;
b) The protein in your cells is degraded, you must add PMSF to inhibit protease activity;
c) Your antibody does not recognize the target protein. Please read the instructions to see if there is a problem.
3. Some of my cell extracts are precipitated and some are very clear, why?
Answer:
a) There may be precipitation because your protein is not completely denatured, you can increase the SDS concentration appropriately, and increase the sample boiling time,
b) It does not rule out that your antigen concentration is too high, then add the appropriate amount of loading buffer.
4. The molecular weight of the protein I made is very small (10KD), how do I do WB?
Answer: You can choose 0.2μml membrane and shorten the transfer time. You can also stack the two films together and transfer them. Just follow the steps.
5. My goal belt is weak, how can I strengthen it?
Answer: You can increase the amount of antigen loaded. This is the most important. At the same time, the dilution ratio of primary antibody can also be reduced.
6. The film background is very dirty, what are the solutions?
Answer: Reduce the amount of antigen loaded, lower the primary antibody concentration, change the primary antibody incubation time, and increase the milk concentration.
7. The target band is blank and there is a background around it. Why?
Answer: The concentration of your primary antibody is high, the catalytic activity of HRP on the secondary antibody is too strong, and your coloring substrate is in the primary
At a critical point, the reaction time is not long, and the surrounding substrate is catalyzed, forming a blank, which is the "anti-bright phenomenon".
Reduce the primary and secondary antibody concentrations, or replace with new substrates.
8. My film is blank, what's wrong?
Answer: If the following problems can be eliminated, most of the problems will occur in the preparation of primary antibodies and antigens.
a) The HRP activity of the secondary antibody is too strong and consumes the substrate;
b) H2O2 in ECM substrate, unstable and inactive;
c) The ECL substrate is not covered to the corresponding position; d) The secondary antibody is inactivated.
9.After developing in the developer for 1 minute and 5 minutes, the film is dark, what is the reason?
Answer:
a) It may be caused by a red light, the film is already exposed, and it can be operated in complete darkness. See if there is improvement .;
b) The development time is too long.
10. Is DAB or ECM better?
Answer: DAB is toxic, but more sensitive, it is the most sensitive substrate for HRP; ECM results are easy to control, but the sensitivity is a bit worse when catalyzed, but if it reaches the threshold, it is particularly sensitive and can detect pg-level antigens. It depends on your experiment.
11. The molecular weight of the antigen detected is larger than the data, what is the matter?
Answer:
a) The antigen formed a dimer. Increase the amount of mercaptoethanol, prolong the boiling denaturation time, can open the dimer.
b) Modification of protein itself such as glycosylation, phosphorylation, etc.
12. The protein can not be transferred to the membrane, but it is on the glue, and the Marker is transferred. Why?
Answer: It may be: a) The sample concentration is too low;
b) The transfer time is not enough.
13. Is it necessary to add NaF etc. when preparing the sample for phosphorylated antibody detection?
Answer: NaF is a broad-spectrum phosphorylase inhibitor, which is generally best added. But it doesn't need to be added, most of the time it is not needed.
14. To verify the presence of the protein on a cell, do I need to do immunohistochemistry and western blot tests? Can the primary and secondary antibodies be shared during these two tests?
Answer:
â‘ Immunohistochemistry can be used for localization, but it cannot be accurately quantified, and sometimes there will be false positives, which is not easy to distinguish from the background; Western blot can specifically detect a certain protein molecule for quantification, but it cannot be localized.
â‘¡ The primary antibodies of the two experiments are sometimes not universal. The company's product description will generally indicate what experiments can be carried out, whether it is suitable for paraffin section or frozen section during immunohistochemistry.
15. When doing Western Blot, the protein was extracted and frozen (without protease inhibitors). The primary antibody used by Dr. Bode was still a bit traced. Now it is getting worse. The sample volume has been increased to 120μg. Santa cloz's primary antibody is still not working. What is the reason? Is it possible to add PMSF to protease inhibitors alone?
Answer: It is suspected to be a sample problem, which may be:
1. The sample cannot be repeatedly frozen and thawed;
2. No protease inhibitor was added to the sample. At the same time, it is recommended to check the Western Blot process to increase the primary antibody concentration. For the addition of protease inhibitors, it is generally sufficient to add PMSF. It is best to add a few more kinds of protease inhibitors.
16. At the cellular level, do Western blot. How many proteins are enough for western blot?
Answer: Generally 5 × 10 ^ 6 is enough
17. Can the same sample extract RNA and protein at the same time? Does this affect western blot?
Answer: Yes, no problem.
18. Can the same protein sample be tested for Western Blot with two factors at the same time?
Answer: Of course, some can even measure dozens of samples at the same time.
19. If the target protein is membrane protein or cytoplasmic protein, what should be paid attention to?
Answer: If it is a membrane protein and a cytoplasmic protein, the detergent used is much milder. It is best to add NaF to inhibit the activity of phosphorylase.
20. The protein content of my sample is very low, less than 1 microgram per microliter, but it is often found that only a part of the protein is transferred to the membrane when the membrane is transferred, that is, all protein strips found in some holes after the membrane is stained The bands are all there, but the color has faded, is there any way to solve it?
Answer: You can increase the sample volume, there is no problem, and you can use the reduced current to extend the time and add 20% methanol when transferring.
21. The protein you want to separate is of 200kd molecular weight. What is the appropriate concentration of separation gel for SDS-PAGE electrophoresis? How much should the concentration of the laminating gel be? Is it easy to use Western Blot for a protein with such a large molecular weight?
Answer: 200kd protein is not easy to do, 7% for separating gel and 3.5% for laminating gel.
22. If the sample volume is overloaded, what method should be used to increase the sample volume?
Answer: You can concentrate the sample, or you can dialyze a part of the small molecule protein according to your target molecular weight. In general, there is no problem with an overload of 30%. If it has exceeded a lot, and the small molecular weight is also necessary, you can consider increasing the thickness of the glue, you can try 1.5mm.
23. How long can the protein be stored after denaturation?
Answer: -80 ℃, no problem for one or two years. The two most critical two: don't be hydrolyzed by proteases; don't be digested by bacteria (also hydrolyzed by enzymes).
24. The molecular weight of the protein I measured is 105KD. It stands to reason that the separation gel should use 7.5%, but the information requires that the separation gel and the concentration gel should use 11% formula. I do n’t know why?
Answer: Both of the gels mentioned above can be used because the 105KD protein is within the linear resolution range of the two gels, but pay attention to the position of the band.
25. The second antibody is a biotinylated antibody, and the third antibody is an avidin biotin system. I wonder if the blocking solution needs to be adjusted after using this scheme, can I use 5% skimmed milk powder again? Skimmed milk powder affects the production of avidin biotin, right?
Answer: Skimmed milk powder cannot be used because skimmed milk powder contains biotin. It should be better to use BSA instead.
26. What is the total amount of protein loaded at one time, is it related to the expression of the target protein?
Answer: Western Blot generally loads 30-100 micrograms. The results are related to the abundance of the target protein, the amount of sample loading, the amount of primary and secondary antibodies and the incubation time, as well as the length of color development time. In order to get a positive result when starting to touch the conditions, each step can be measured a little longer, and of course the background comes out. To get good results, it is easier if the antibody is good, and you need to try again and again if the antibody is not good. Of course, some are not suitable for Western Blot.
27. How to deal with samples when doing western tissue samples?
Answer: Grinding, homogenization, and ultrasonic treatment must be performed. The protein solubility will be better. Centrifugation should be sufficient. Membrane proteins need to be extracted more vigorously. Low-abundance membrane proteins may have to be extracted stepwise (ultracentrifugation). Another point is that the protease activity in the tissue is stronger, and it is necessary to pay attention to inhibit the protease activity (add PMSF).
28. Ask large molecular weight protein 200KD, what should we pay attention to when doing western?
Answer: When doing Western Blot with 200kd protein, you should pay attention to it. It is better to choose the separation gel> 7%; be careful when peeling the gel; the transfer time needs to be extended accordingly; make reference to the molecular weight (otherwise, if there are miscellaneous bands, do not know how to analyze).
29. Is there any way to increase the sample load?
Answer:
a) The sample can be concentrated; increase the sample volume to increase the sample volume;
b) Dilute and denature with 5 times loading buffer
30. Are there any specific requirements for protein loading?
Answer: The sample volume should be determined according to the requirements of the experiment. If the requirements are quantitative and semi-quantitative Western Blot, the sample volume should be equal. If it is only to be qualitative, there is not much relationship. Try as much as possible, but do not overload.
31. Is the ratio of primary antibody to secondary antibody important?
Answer: It is more important. Adjusting the ratio of primary and secondary antibodies can remove some non-specific background.
32. What are the requirements for the secondary antibody to phosphorylate a factor Western Blot?
Answer: There is no requirement for secondary antibodies. It depends on your experimental conditions. It is generally recommended to use HRP-labeled secondary antibodies.
33. Can I use the same antibody for immunohistochemistry and Western Blot?
Answer: During immunohistochemistry, antibodies recognize unmodified antigenic determinants (also known as epitopes), some epitopes are linear, and some are conformational; linear epitopes are not affected by protein denaturation. Both protein and boiled protein are contained; conformational epitopes are restricted by the spatial structure of the protein, and denaturation will disappear after cooking. If the antibody you use recognizes several consecutive amino acids on the protein, that is, a linear epitope, then this antibody can be used for both immunohistochemistry and Western, and if the antibody recognizes a conformational epitope, it can only be used for Immunohistochemistry.
34. Can the antibodies in Western Blot be used repeatedly?
Answer: Antibody working solutions are generally not recommended for storage and repeated use, but if the antibody is more precious, it can be used repeatedly 2-3 times. It should be used within 2-3 days after dilution and stored at 4 degrees to avoid repeated freezing and thawing.
35. When doing Western Blot, what is the purpose of soaking the PVDF membrane with methanol?
Answer: The purpose of the methanol bubble in the PVDF membrane is to activate the positively charged groups on the PVDF membrane, making it easier to combine with negatively charged proteins. It is also the purpose of adding more methanol when doing small molecule protein transfer.
36. Can the phosphorylated and non-phosphorylated antigens of the same antibody be on the same membrane?
Answer: Yes.
37. The bands stained with Ponceau after transfer of the membrane, why the transfer membrane at one end of the large protein molecule does not seem to be very good, why?
Answer: This is normal. The protein transfer of large molecules is slow. If you extend the transfer time and current, the end of the large molecule will be much better, but the small molecule may become lighter.
38. When doing Western Blot, the same antibody immunohistochemistry can be made, but Western Blot cannot?
Answer: This is mostly a question of antibodies, depending on the description of the antibody, whether it can do Western Blot and immunohistochemistry.
39. If it is a 6 × 8 transfer film, how much primary antibody should be added?
Answer: The dilution of the primary antibody is stated. You will know based on your primary antibody, but the incubation volume of such a large membrane is generally at least 3-5ml.
40. What are the requirements of the buffer solution in the upper and lower tanks and how can the best results be achieved.
Answer: No special requirements. But we usually put fresh buffer in the upper tank, and the lower tank can be the buffer that has been used once or twice
41. What is the reason why the glue always "shrinks" when I run electrophoresis? Is there some wrong composition?
Answer: No problem, the water in your glue is evaporated. Wrap the plastic wrap overnight and add some water to maintain humidity; it may also be a problem with the mother liquor (30% polyacrylamide), you can reconstitute an observation; replace the reagents, try to replace them, choose good Reagents.
42. The molecular weight of the protein has a large span. If the separation is as small as 21KD, medium to 66KD, and as large as 170KD, can it be done at once?
Answer: Such a wide distribution is not easy to transfer. It is generally recommended that 21kd and 66kd can be transferred together, 12% SDS-PAGE, wet transfer 120mA, 45-60min. It can be adjusted according to your laboratory experience; 170kd uses 7% SDS-PAGE, 200mA 90-120min.
43. Can't transfer large molecular weight protein to the membrane well, how to solve the problem of low transfer efficiency?
Answer: Consider: adding 20% ​​methanol (referred to the final concentration) to the transfer buffer, because methanol can reduce the protein elution efficiency, but can increase the binding capacity of protein and NC membrane, methanol can prevent gel deformation, methanol is high The molecular weight protein can extend the transfer time; the final concentration of 0.1% SDS in the transfer buffer is also to increase the transfer efficiency; use a high-quality transfer membrane, or use a small pore NC membrane (0.2 microns).
44. I use a visual marker, but the electrophoresis does not run 8 bands at all. What is the reason? How to improve it? Used 8%, 10%, 12% of the glue. marker is new.
Answer: Generally speaking, it is the small molecular weight Marker that runs away, try to increase the gel concentration or reduce the electrophoresis time. Of course, gradient glue is also a good choice.
45. Is it necessary to add ACTIN internal reference for Western Blot semi-quantitative experiment?
Answer: It is best to add internal reference to the experiment of publishing articles, and the experiment is rigorous.
46. ​​What is the appropriate choice of internal reference for nuclear antigen Western Blot?
Answer: Histones can be selected. The expression of histones in the nucleus is very stable. Many of them can be used as internal controls. Checking online can select the internal control you want.
47. Do semi-quantitative Western Blot, internal reference β-actin, GAPDH which is better?
Answer: Just use beta-actin.
48. I would like to ask what are the principles when selecting protease inhibitors in cell lysates? Are they not affected by tissue sources? Is there a difference between membrane and cytoplasm?
Answer: Generally speaking, it is sufficient to add a broad-spectrum protease inhibitor during extraction, and keep the temperature low during operation. Unless there is a literature that specifically indicates the use of special methods, there is generally no difference.
49. The skimmed milk powder sealing solution after film transfer is 5% TBST skimmed milk powder ", where is the last T in TBST Tween, and what is the concentration?
Answer: It is Tween, the formula is as follows: Tris-Buffered Saline Tween-20 (TBST), NaCl: 8g, Tris base: 2.42g, add 800ml of water to dissolve, add 500-1000ul of Tween-20, adjust the pH to 7.4 with HCl, add water to determine Capacity to 1L.
50. Are there any regulations on the temperature of the closed, primary and secondary antibodies, for example, at room temperature, or at 4 degrees?
Answer: Both can be carried out at room temperature. If the time is not enough, the primary antibody incubation can be carried out at room temperature for one hour and then overnight at 4 degrees.
51. What is the principle of PVDF membrane and nitrate membrane binding protein?
Answer: Generally speaking, nitrocellulose membranes are associated with proteins by hydrophobic action. In this case, after repeated washing for several times, the proteins will fall off easily, resulting in poor results. The PVDF membrane mainly connects with the protein through its positive charge, and also has a hydrophobic effect, but it is relatively weak. In this case, the PVDF membrane and the protein are more firmly bonded and not easy to fall off, and the result is better.
52. How can I make the glue run very beautiful, the swim lanes can be very straight, is the amount of sample loading and glue filling important, and what are the voltage requirements?
Answer: There are several factors that affect the quality of rubber running: 1. Voltage, small voltage will make the molecular sieve effect of the rubber fully exerted. The lower the voltage, the more beautiful the strip. The concentrated gel 55v and the separation gel 75v can run very well. 2. The uniformity of the glue, the more uniform the glue, the narrower the strip, the more uniform the separation. Before pouring the glue, be sure to mix it well. The glass plate must be clean. When double-distilled water is isolated, it must be added relatively lightly to avoid diluting the upper separation glue.
53. Why is there a loss of small molecular weight proteins when increasing the transfer of large molecular weight proteins? What are the reasons?
Answer: During the transfer of small molecules, proteins will pass through the membrane, so after the large molecules go up, some of the small molecules will pass through.
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