I'm working on a project where I need to reliably quantify changes in NRF2 / NFE2L2 expression in mouse fibroblasts. Meanwhile, a colleague in my lab would like to measure NRF2 levels in mouse livers.
We have tested Cell Signalling's NRF2 monoclonal (cs-12721), but it gives very low signal. We've had to use a high-performance ECL substrate to get any usable signal from the blots, but in such cases the blots suffer from *very* high background which does not wash off even under high salt (500mM NaCl) or mild detergent (0.2% SDS). Other ABs (e.g. GAPD, cs-5174) give excellent bands with these samples. (IMAGES BELOW)
I'd like to test other ABs, but thought I'd get RG's opinion before I waste money on some worthless polyclonals.
Added: cell lysates were loaded at ~30ug/lane. Liver lysates were loaded at ~50ug/lane. Ponceau S and Coomassie images available on request.
Hi Brian
It is the common obstruction in estimating Nrf2 levels using monoclonal antibodies as the background levels are very low. I would suggest you to use Anti-Nrf2 antibody from abcam which i found far better then CST make in this particular case. I am sure it would help your cause.
Thanks for the quick reply. It looks like Abcam has several NRF2 ABs. Can you tell me which one you've had luck with? I had been considering testing the Abcam Rabbit mAb, but will test one of their polyclonals if you've had luck with it. The link below will go to a list of all of the primary, unconjugated rabbit ABs against total NRF2 that Abcam offers.
Added: I found that a co-worker has some of this Abcam NRF2 AB (http://www.abcam.com/nrf2-antibody-ab31163.html) on hand, which I am testing presently.
http://www.abcam.com/products?keywords=NRF2&selected.classification=Primary+antibodies&selected.targetName=Nrf2&selected.modification=Unmodified&selected.conjugate=Unconjugated&selected.hostSpecies=Rabbit
No dice. I followed up cs-12721 by re-blotting with ab31163 (no stripping). In this blot the main bands detected in cell lysates are at ~80 kDa and ~60 kDa, as opposed to the expected mass of 100 kDa.I believe that most of the signal shown here is from ab31163, as under similar exposure conditions the signal from cs-12721 is barely detectable. This blot was imaged using Pierce ECL. I guess the Abcam monoclonal might be the next best option.
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