Hello everyone,
Recently, I detected the expression of p-p65 ( HY-P80470, MCE) and p-IKBα ( HY-P80466, MCE) in HSF cells by Western blot , but the results seem problematic—no bands appeared, and the background is blurry. What could be the reason?
The entire membrane shows blurry bands for this product. Possible reasons include:
Recommendations for improvement:
Based on the current experimental results, it is difficult to determine whether the bands are too intense. You may try adjusting the primary antibody dilution to 1:1000.
HY-P80470 is a phospho-specific antibody. The molecular weight we display is the theoretical molecular weight. Below is our validation image. Due to phosphorylation modifications, an increase in molecular weight may be observed. The apparent molecular weight may also deviate slightly when using pre-stained markers because of buffer effects, but in your case, the band appears significantly larger. We recommend referring to our validated cell lines and treatment conditions to determine whether the band around 50 kDa corresponds to your target.
Additionally, for this antibody, we recommend using a membrane with a 0.44 μm pore size.
Some non-specific binding has also been observed with this antibody. You may also try adjusting the primary antibody dilution to 1:1000.
When the antibody concentration is too high, there is an increased likelihood of the antibody binding to non-specific sites on the membrane, which can result in elevated background signals. Excessive accumulation of antibody on the membrane can also make it difficult to distinguish specific bands from the background.
The answer to this question comes from MCE Technical Support.
- Badges
- Science topic
- Similar topics
- Computer Communications (Networks)
- Simulators