I am using a Nrf2 Antibody (A-10): sc-365949 from santacruz which is a new replaced monoclonal antibody. With this antibody, I am getting an intense single band in my control sample at around 50-60kda.
But after treatment of my drug of interest gives extra clear band at 110 kda. The most interesting thing is with dose treatment of drug the 50-60kda band decreases while at higher drug treatment 110kda intensifies.
Many papers have their different views in Nrf2 molecular weight and true Nrf2 band in western Blot. Here, I just needed to know which band, I should consider for Nrf2 expression and if 50-60kda band is non-specific band, then why it is decreasing with the treatment and if not, why one band is decreasing and other one (110kda) is increasing.
Please help in this regard.
You may ask the company why they offer an antibody that detects a 60kDa band when Nrf2 band has a mobility of approx 100 kDa even when the MW is 60 kDa. I would use an antibody that has been better characterized in addition to this new one (which I imagine you are using because it is free). An nrf2 shRNA/CRISPR or a nrf2 KO tissue lysate would be ideal as control.
This paper has an excellent Nrf2 antibody testing and discussion.
Article Comparison of human Nrf2 antibodies: A tale of two proteins
first, thank u so much for your suggestion. I have followed your suggested article too. But available literature of my control cells (cervical cancer) supports this santacruz antibody (older one with 60-68kda band), where they validated high expression of Nrf2 and they have shown down-regulation of Nrf2 (60-68kda band) expression with use of shRNA or siRNA.
In my samples, I am able to get this 60-68 kda band in low dose treatment and with high dose of treatment 110 kda band (clean band) appears.
What could be d reason?
Dear Suchitra,
I am actually not ansewering your question but asking something else... I am trying to use the same antibody sc-365949 to detect NRF2 expression in my primary fibroblasts upon oxidation stress. However, I am not observing any band at all. Could you tell me if you are doing any special extraction protocol? I have already used RIPA and SETH buffer. Thanks in advance!
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