1. Is higher ECL luminous sensitivity better?
No. Most of the experiments have protein concentration at the pg-ng level, and it is fine to choose the appropriate luminescent solution.
2. Why do I need to keep ECL away from light?
Because luminal and other luminescent components in ECL are easily inactivated by light, so they need to be stored away from light.
3. The signal of target protein is weak or absent.
One of the common problems in Western blot luminescence assay is that the signal of target protein is weak or absent. Firstly, consider whether the membrane transfer is insufficient/over-transferred, if so, optimize the membrane transfer conditions; secondly, under the premise of transferring the membrane appropriately, consider whether the antibody-antigen sensitivity is too low, or the substrate HRP or substrate activity is reduced, which can be solved by increasing the volume of samples/increasing the concentration of antibody/using a sensitive substrate/lengthening the time of pressing the film. A short exposure time can also reduce the protein signal, so extend the exposure time of the film.
4. High background.
Deep background is the most common problem in Western blot luminescence detection, and there are many reasons for the deep background. First, the problem of closure, closure conditions are generally 5% milk or BAS room temperature closure 2-4h, but preferably 4 overnight; in fact, milk is good for most antibodies, but the secondary antibody and milk may have cross-reactivity, so washing is important. In addition, BSA has a single protein, which reduces the background caused by other components of milk. Secondly, incomplete elution of TBST can also lead to dark background; increasing elution time, frequency and dosage appropriately can reduce the background color. Thirdly, the deep background of the strip is also related to the exposure time, the exposure time is more than half an hour the background is very normal, so it is recommended to expose for 1-10min if possible. westernblot luminescence reagent EnlightTM produced by Ingunn contains unique luminescence enhancer and precise luminescence substrate, which has higher sensitivity and stability than ordinary luminescence reagent, and the luminescence time is long and the background is lower. The background is lower. Fourth, if the antigen-antibody concentration/HRP is too high, it will directly or indirectly bind to the membrane and cannot be eluted off, etc. Consider lowering the antibody concentration or the amount of protein sample.
5. Non-specific bands
Non-specific bands are associated with antibody cross-reactivity. Monoclonal antibodies have better specificity and higher purity than polyclonal antibodies. In addition, appropriate dilution of primary/secondary antibody concentration is necessary. If degradation of the sample occurs during processing, the sample is processed with a protein inhibitor operated on ice.
6. The band on the membrane turns yellow or white after development.
The sensitivity or background may be too high due to too high HRP and the antibody concentration should be reduced.
7. There are white spots within the bands that do not develop.
This is related to the membrane transfer and development operation. Try to equilibrate the membrane before transferring and take care to avoid air bubbles between the gel and the membrane.
8. scattered small round spots
It is caused by the impurity particles in the milk sealing solution, to solve this kind of problem, you can configure the sealing solution in advance and store it at 4 degrees Celsius, don't mix it when you use it, and only use the upper layer.