How much primary antibody and secondary antibody should be used in WB experiments?
A: Depending on the potency, concentration and affinity of different antibodies, the dilution ratio of most commercial antibodies is around 1:200-1:2000; the concentration of secondary antibody is usually around 1:5000-1:0000.
It is recommended to do pre-experiment first, you can refer to the antibody instructions for pre-experiment first, and adjust according to the results of pre-experiment.
Can antibodies from WB experiments be generalized to other experiments such as IHC?
A: During WB, the target protein is denatured by denaturing agents such as SDS and mercaptoethanol, and the antigenic epitopes inside the protein are exposed, and the antibody recognizes the linear antigenic epitopes of the target protein. Therefore, most of the antibodies used in IHC, IP, FACS and other experiments can be used in WB experiments.
Why does the size of the target band sometimes deviate from the theoretical expected molecular weight?
A: When the actual size of a band deviates from the theoretical one, it may be due to a number of reasons.
- The band shifts up when the protein undergoes post-translational modifications such as phosphorylation, glycosylation, etc.
- The band may shift down when the protein is sheared (e.g., precursor), or there may be two small target bands.
- Proteins have different variable shears or isoforms.
- Protein denaturation is incomplete in the presence of strongly interacting macromolecules.
- The pre-stained protein marker used in WB experiments may also deviate from the theoretical expectation of apparent molecular weight due to the unstable degree of dye carried.
Why do stray bands sometimes appear?
A: Possible reasons include insufficient antibody specificity, different modification states or shear degradation of the target protein, excessive use of primary or secondary antibodies, excessive protein loading, long exposure time, insufficient membrane rinsing, etc. The appearance of spurious bands can be minimized by optimizing the conditions. However, when a small amount of stray bands are present due to antibody or sample characteristics, it does not affect the application of the data image as long as it does not affect the band discrimination of the primary objective.
Why does the film sometimes appear to have a significantly darker background?
A: In addition to the experimental reasons such as insufficient closure, impurities in the sample, excess antibody, insufficient rinsing, etc., when the signal of the target band is too weak for specific identification, the exposure time has to be extended to show the target band, which subsequently makes the background dirty. In this case, the "sex-to-noise ratio" of the whole reaction is too low, and it is better to use a primary antibody with better specificity and affinity.
Why is it sometimes done without a destination band?
A: Possible reasons.
- The expression of the target protein contained in the sample is too low or no expression, so confirm the feasibility of the sample.
- The protein was degraded during the extraction process. Keep the protein extraction at a low temperature and add the appropriate protease inhibitor.
- The concentration of antibody incubation is too low or the time is too short. The amount of antibody incubation should be optimized, and overnight incubation at 4°C is recommended for primary antibodies.
- Antibodies are inactivated and should be stored correctly.
My destination band is weak, how can I strengthen it?
A: The most important thing is to increase the amount of antigen on the sample, also the primary antibody dilution ratio can be adjusted upwards.
Why is the target band blank and surrounded by background?
A: The concentration of primary antibody is high, and the catalytic activity of HRP on secondary antibody is too strong. The chromogenic substrate is at a critical point, and the reaction time is not long enough to finish catalyzing the surrounding substrate, forming a blank.
Improvement method: Reduce the concentration of primary antibody and secondary antibody, or replace the newly imported chromogenic substrate.
How to do WB with very small molecular weight of protein (around 10KD)?
A: Try to choose a 0.2 μm membrane to shorten the transfer time. You can also stack two membranes together and then electrotransfer them.
Why are the strips in smiley face shape?
A: The gel is not cooled uniformly, the middle cooling is not good; the temperature of electrophoresis system is high, and the electric field intensity is too large.
Why are the strips in frown shape?
A: It may be due to improper device, or there are bubbles at the bottom of the gel and glass baffle, or incomplete polymerization on both sides.
Why are the strips trailing?
A: The sample is poorly solubilized or there is some degree of protein degradation.
Why are there textures (longitudinal stripes)?
A: The sample contains insoluble particles and may have had problems with protein extraction.
Why are the strips skewed?
A: The electrodes are not balanced or the sample is skewed.
Why do the strips spread on both sides?
A: Too much sample was added and diffused around the wells. Reduce the sample volume or use 5X loading buffer.
To do WB at the cellular level, how many cells extract enough protein to do WB?
A: Generally, 5×106 is enough. Special cases need to be determined according to your own research.
Why is my film blank?
A: Exclude the following problems before considering whether there is a problem with the primary antibody and antigen preparation
a) The HRP activity of the secondary antibody is too strong and the substrate is consumed
b) The ECM substrate contains hydrogen peroxide, which is unstable. It is easy to deactivate when exposed to light or high temperature.
c) ECL substrate is not covered to the corresponding position.
d) The time of secondary antibody is expired, or the secondary antibody is inactivated due to improper use in general.
What is the cause of the negative being dark after I have developed it for 1 minute and 5 minutes in the developer?
A: a) It may be caused by the red light, the film was originally exposed, you can operate in complete darkness to see if there is improvement
b) The development time is too long, usually 3-5 minutes is enough, special cases need to find out the color development time.
Can the same protein sample be subjected to WB assay of two factors at the same time?
A: Of course, some can even measure dozens of samples at the same time. If the antibody specificity is high and high potency, using 2 primary antibodies at the same time, 2 different proteins can be detected on one membrane.
If the target protein is a membrane protein or a cytoplasmic protein, what should I pay attention to?
A: If it is membrane protein and cytoplasmic protein, the detergent used is much milder, and it is better to add NaF to inhibit the phosphorylase activity. Some membrane proteins are not easy to extract, especially the subcellular organelle membrane proteins. You can consider the latest invent column extraction method, which has high extraction efficiency and less protein loss.
