Hi everyone,
I’ve been working with NF-κB p65 (HY-P80765) in Western blot (WB) experiments, but I’m consistently observing multiple bands or an increased molecular weight for p65. This is occurring even though I’m using a well-validated antibody.
Is this due to post-translational modifications (like phosphorylation, acetylation, or ubiquitination), alternative splicing, or possibly proteolytic cleavage? Could the issue be related to protein degradation, antibody cross-reactivity, or sample preparation?
Has anyone else encountered this issue with p65 detection? Any suggestions on how to better interpret these bands or ways to prevent them?
Thank you in advance for your insights!
Hi, Janice. The appearance of multiple bands or an apparent increase in molecular weight of NF-κB p65 in Western blot analysis is mainly due to post-translational modifications (PTMs) and the existence of distinct isoforms.
NF-κB p65 undergoes various PTMs, including phosphorylation, acetylation, ubiquitination, and nitrosylation, which alter its electrophoretic mobility and result in shifts from the predicted molecular weight. In addition, alternative splicing and newly identified p65 isoforms have been reported, which can also contribute to the detection of multiple bands.
Therefore, the observation of multiple bands is not necessarily an artifact but rather reflects the complex regulation of NF-κB p65 through PTMs and isoform diversity.
Reference:
1. Kwon JW, et al. Activating transcription factor 3 represses inflammatory responses by binding to the p65 subunit of NF-κB. Sci Rep. 2015 Sep 28;5:14470.
2. Leeman JR, et al. Alternative splicing in the NF-kappaB signaling pathway. Gene. 2008 Nov 1;423(2):97-107.
3. Spinelli G, et al. A new p65 isoform that bind the glucocorticoid hormone and is expressed in inflammation liver diseases and COVID-19. Sci Rep. 2021 Nov 25;11(1):22913.
The answer to this question comes from MCE Technical Support.
- Badges
- Science topic