Western blot precautions and frequently asked questions:
1 Some of my cell extracts have precipitation, some are clear, why? A: There may be precipitation because your protein is not denatured completely, you can increase the concentration of SDS appropriately, and at the same time, extend the boiling time of the sample; it is not excluded that your antigen concentration is too high, then add an appropriate amount of sampling buffer.
2 The molecular weight of my protein is very small (10 KD), how to do WB? A: You can choose 0.2 μm membrane and shorten the transfer time. You can also stack two membranes together and transfer them.
3 Is it better to use DAB or ECL for final color development? A: DAB is toxic, but more sensitive, it is the most sensitive substrate for HRP; ECL is easy to control the result, but the sensitivity is a little bit worse when it is catalyzed, but if it reaches the threshold value, it is especially sensitive, and it can detect the pg grade protein, according to the situation of your experiment.
4 Do I need to do immunohistochemistry and western blot to verify the presence or absence of the protein on a cell? Can the primary antibody and secondary antibody of these two tests be used together? A: ①Immunohistochemistry can be used for localization, but not accurate quantification, and sometimes there will be false positives, it is not easy to distinguish from the background; Western blot can be used to specifically detect a protein molecule, quantification, but not localization. ② The primary antibody of the two experiments sometimes can not be used universally, the company's product description usually explains what experiments can be carried out in immunohistochemistry, whether it is suitable for paraffin sectioning or frozen sectioning.
5 Cell level to do western blot, how many cells to extract enough protein to do western blot? A: Generally 5×106 is enough.
6 Can RNA and protein be extracted from the same sample at the same time? Will this have any effect on the western blot? A: Yes, there is no problem.
7 Can the same protein sample be subjected to Western Blot for two factors at the same time? A: Of course you can, some can even measure dozens of samples at the same time.
8 How long can the protein be stored after denaturation? A: -80℃, no problem for one or two years. The most critical two: don't be hydrolyzed by protease; don't be digested by bacteria (also hydrolyzed by enzyme). 9 The secondary antibody is biotinylated antibody, and the tertiary antibody is affinity biotin system, I wonder if the containment solution should be adjusted after adopting such a scheme, and whether 5% skimmed milk powder can be used again? A: Skimmed milk powder cannot be used because it contains biotin, it should be better to use BSA instead.
10 How to handle the samples when doing western of tissue samples? A: It is necessary to grind, homogenize, and sonicate the samples for better protein solubility, centrifugation should be sufficient, membrane proteins need to be extracted more vigorously, and low abundance membrane proteins may also need to be extracted in steps (ultracentrifugation). Another point is that proteases are more active in tissues and care needs to be taken to inhibit protease activity.
11 Can the same antibody be used for immunohistochemistry and Western Blot? A: Immunohistochemistry antibody recognizes undenatured antigenic determinants (also known as epitopes), some epitopes are linear, while others are conformational; linear epitopes are not affected by protein denaturation, and are contained in both natural proteins and boiled proteins; conformational epitopes, due to limitations in the spatial structure of proteins, will disappear after boiled denaturation. If the antibody you use recognizes several consecutive amino acids on the protein, a linear epitope, then this antibody can be used for both immunohistochemistry and Western, whereas if the antibody recognizes a conformational epitope, it can only be used for immunohistochemistry.
12 What is the purpose of soaking PVDF membrane in methanol when doing Western Blot? A: The purpose of soaking the PVDF membrane in methanol is to activate the positively charged groups on top of the PVDF membrane, so that it is easier to bind with negatively charged proteins, and this is also the purpose of adding more methanol when doing small molecule protein transfer.
13 Can the phosphorylated and non-phosphorylated antigens of the same antibody be detected on the same membrane? A: Yes.
14 The molecular weight of the protein spans a large range, such as to separate the small 21KD, medium to 66KD, large to 170KD, can it be done at once? A: Such a wide distribution is not good transfer, general advice: 21KD and 66KD can be transferred together, 12% SDS-PAGE, wet transfer 120mA, 45-60min can be, can be adjusted according to your laboratory experience; 170KD with 7% SDS-PAGE, 200mA 90-120min.
15 Are there any principles for selecting protease inhibitors for cell lysates? Is it affected by the origin of the tissue? Is there a difference between cytosol and cytosol? A: Generally speaking, it is enough to add broad-spectrum protease inhibitors at the time of extraction, and keep low temperature during operation. If there are special requirements, you can choose the appropriate protease inhibitor.
16 What is the principle of binding protein by PVDF membrane and nitrate membrane? A: Generally speaking, nitrocellulose membrane is linked with protein through hydrophobic effect, in this case, after repeated washing several times, the protein is easy to fall off, and the result is poor, PVDF membrane is mainly through the positive charge on its membrane and protein bonding, and also has a hydrophobic effect, but it is relatively weak. In this case, PVDF membrane and protein are more firmly bonded, not easy to fall off, and the result is better.
17 What is the appropriate choice of western blot internal reference? A: This is related to the expression location of the target antigen. For cytoplasmic and whole-cell proteins, we can choose β-actin, GAPDH and Tubulin; for mitochondrial proteins, we can choose VCDA1/Porin and COXIV; and for intranuclear antigens, we can choose Lamin B1, TBP and histone.
18 Can't transfer large molecular weight proteins to the membrane well and the transfer efficiency is low? A: Add 20% methanol (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 the gel from deformation, methanol can prolong the transfer time for high molecular weight proteins; add the final concentration of 0.1% SDS to the transfer buffer, which can also increase the transfer efficiency; use glutaraldehyde to cross-linking; reduce the concentration of the gel, e.g., as low as 6-7% or increase the transfer voltage/transferring membrane. 7% or increase the transfer voltage/current to increase the transfer time.
19 Gel swelling or curling during membrane transfer? A: The gel can be put into the transfer buffer to soak for 5-10min before transferring.
20 The strip of running gel is skewed or drifting? A: Electrotransferometer long-term use of sponge thinning, "sandwich" structure is not compact, can be replaced or between the two sponges with a little ordinary straw paper.
21 There are single or multiple white spots on the film after transferring? A: Make sure there are no air bubbles between the film and the adhesive block when transferring the film.
22 The buffer is too hot? A: The concentration of ions in the buffer is too low, the current or voltage is too high, pay attention to cooling down in the process of transferring the film.
23 After developing the film background is too high? A: The PVDF film is not completely wet with 100% methanol before transferring; the film is not washed sufficiently, the number of times the film is washed can be increased; the closure is incomplete, choose the appropriate closure solution and increase the closure time; the concentration of the secondary antibody is too high, reduce the concentration of the secondary antibody; the specificity of the primary antibody is not strong, change to a monoclonal antibody of strong specificity; the exposure time is too long, reduce the exposure time.
24 No bands or very light bands, weak hybridization signal? A: The sample does not contain the target protein or the content is too low, can increase the amount of protein on the sample, set a known standard amount of protein control; antibody dilution ratio is too low, the incubation time is not enough; the transfer membrane is not good, after the transfer of the membrane can be used to watch the staining effect of Lichun red staining; the exposure time is too short, increase the exposure time; the antibody is not properly preserved, the potency of the antibody is lowered.
The position of the 25 purpose bands is on the low or high side? A: The concentration of gel is not suitable, high molecular weight should use low concentration gel, small molecular protein should use high concentration gel.
26 What are the non-specific bands? A: The target protein has multiple modification sites (phosphorylation sites, glycosylation sites, acetylation sites, etc.), which can present multiple bands; the specificity of the primary antibody is not good, use a monoclonal antibody with better specificity; the results caused by the non-specificity of the secondary antibody, you can set up a group of parallel control without adding primary antibody and only secondary antibody to detect whether the secondary antibody has non-specific binding; the sample proteins may be degraded, so pay attention to the operation on the ice when extracting proteins, and add appropriate protease inhibitors. Sample proteins may be degraded, when extracting proteins, pay attention to the operation on ice, add appropriate protease inhibitors, and avoid repeated freezing and thawing of samples; the concentration of antibody is too high, reduce the concentration of primary antibody and secondary antibody.
27 The film is blank, what's wrong? A: If the following problems can be eliminated, then the problem most likely occurs in the primary antibody and antigen preparation. The HRP activity of the secondary antibody is too strong and consumes the substrate; H2O2 in the ECM substrate is unstable and inactivated; the ECL substrate does not cover the corresponding position; the secondary antibody is inactivated.
28 The target band is white with background around it? A: The content of target protein is too high; the concentration of primary antibody is high, and the catalytic activity of HRP on secondary antibody is too strong.