The product data sheet gives the main band at 57 kDa, but I have 4 main bands, one at about 100 kDa, two that could be 57kDa (I ran a mini-gel so it's hard to differentiate), and one below 50kDa. I ran a 10% gel, with about 30ug protein.
When I detected Nrf2 protein, I just found one strong band approximately 55~60 kDa.
And followings are procedure which was underwent.
1. load 16ug protien and running with 12.5%
2. transfer 25V for 90min
3. block with 5% skim milk for 1 h at room-temperature (RT)
4. incubated with 1'Ab:5% skim milk=1:1000 for 1 h at RT and O/N at 4'C
5. incubated with 2'Ab:5% skim milk=1:5000 for 1 h at RT
I'll happy that my procedure was helpful to you.
And I wonder there was poly-ubiquitinated Nrf2 which was detected around 100kDa.
Thank you so much. I'm thinking there might be poly-ubiquination involved. Also, I'm running cytosol and nuclear extracts as opposed to whole cell lysates and am getting two very strong bands of slightly different molecular weights. Do you or does anyone else know if Nrf2 should be slightly heavier in the cytosol versus the nucleus?
Antibodies from Santa Cruz are notoriously bad. I would definitely run the suggested controls before making any assumptions about protein modification.
Hi everyone,
I have the same problem (if you can call it that) , my bands are usually in the 100 kd region. There are however some groups that say it's supposed to be this way, for instance in the following letter:
http://online.liebertpub.com/doi/pdf/10.1089/ars.2012.4754
Interestingly the authors find Nrf2 to be at the ca. 100kd point also when the protein is expressed in E. Coli, so I guess that rules out ubiquitination. They theorize that runs weirdly because it containes a whole bunch of acidic residues. I don't know about that though.
At any rate, with knockdown/induction etc. I see that the most interesting things happen at the 100 kd band.
Hope this helps,
Hendrik
P.S. As far as Santa Cruz is concerned I would like to express agreement with others in this thread.
Thank you all so much.. I agree, Santa Cruz is not satisfactory so far. I do prefer cell signal.. but SC did send us a free monoclonal (full size!) Nrf2 when I contacted customer service, so they really are trying..
GeneTex is another good option for finding antibodies. They do a lot of validation work.
Hendrik, thanks for your links to that papar. I have been experiencing the same problem as you are. Actually, I found another paper pointing out the 96KD form of Nrf2 is due to acidic amino acid in Nrf2 drags this protein behind its theoretical MW, and the 98KD and 118KD is due to phosphorylation by casein kinase.
I find that sc-722X Nrf2 antibody only works for ChIP but it definitely ChIPs Nrf2-responsive genes. Perhaps, Santa Cruz is not all that bad after all.. But it does not work reliably for WB unless you have an Nrf2 KD/KO on the side.. Has anyone tried that for the mouse/human samples? I'd be curious to compare the results..
Hi all, we found several bands with sanza cruz C-20 clone as well. Maybe the cellsignaling XP antibody might provide an alternative:
http://www.cellsignal.com/products/12721.html
Those XP antibodies are usually well validated. However, I still find the "96-kDa -due-to-acidic-amino-acids" theory not convincing. Proteins with many acidic residues carry more negative charges under SDS-PAGE conditions and should, thus, should run FASTER than proteins lacking such residues (please tell me if I'm wrong at this point). 96 kDa bands instead of predicted 66 kDa bands mean that this protein runs SLOWER than expected.
I have serious concerns that any of the abovementioned antibodies really recognizes Nrf2.
Has anyone done a mass spec analysis of the respective bands yet?
Cheers,
Simon
Nrf2 antibody from sc722 gave multiple bands between 250-150kDa (1 band), 150kDa(1 band), 150-100kDa (3 bands),75 kDa 1 band) , 50-37 kDa (3 bands) and 37-25kDa(4 bands). To check for the specific Nrf2 band we treated with cells Nrf2 inducer tbhq 1mM 4hrs but we got very little increase in band intensity really difficult to make out using the most sensitive femto kit from Thermoscientific. All these were done using nuclear lysate and 50ug sample was loaded . Whether the method for lysate preparation we did with Thermoscientific NE-PER Kit is not suitable for this protein. Can anyone tell me the best way to isolate this protein?
There is a lot of confusion regarding Nrf2 and it's observed molecular weight using Western immunoblotting. Unfortunately many published papers have based their conclusion on wrong signal, the lower 55-68kDa band.
Donna Zhang's lab has investigated Nrf2 migration in Western immunoblotting thoroughly and their conclusion was that Nrf2 is observed at 95-110 kDa (Lau A et al., 2013; Antioxid Redox Signal. 2013 Jan 1;18(1):91-3. doi: 10.1089/ars.2012.4754).
We have tested both the C-20 Santa Cruz antibody and Cell signaling (#12721) antibody. Although the Santa Cruz antibody gives several bands (not suitable for microscopy!), with a strong signal around 60-68 kDa, it detects a band close to 100 kDa, which is the correct size. The C-20 antibody is better in detecting the 100 kDa band compared to the Cell signaling antibody (usually we prefer to use antibodies from Cell signaling). The C-20 antibody has also been convincingly confirmed to detect Nrf2 by the Donna Zhang lab. Please note that Santa Cruz actually describe a 55-68 kDa band in their data sheet for the C-20 antibody, whereas Cell signaling present a 100 kDa band. It is not at all strange that there are a lot of confusion here!.
Poly-ubiqutination of Nrf2 is NOT the reason for the difference between observed and predicted MW, as suggested in this discussion. Poly-ubiqutination would most likely give a smear, not a single distinct band much higher than the theoretical predicted MW.
The difference in MW weight for Nrf2 is explained by high content of acidic amino acids. It has been suggested in this discussion that high content of acidic amino acids will give the opposite result - an observed lower molecular weight. This is not true.
Please see Guan Y et al., 2015; Sci Rep. 2015 Aug 27;5:13370. doi: 10.1038/srep13370. In this paper they have compared several proteins with high content of acidic amino acids, all of which show reduced migration in Western immunoblotting.
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