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I have a question concerning the antibody against NFE2L2. I attached two pictures of Western Blot using the NFE2L2 antibody on a SDS PAGE of whole mouse tissue extracts. As you can see, there are many bands. The one that is expected should be 68 kDa, then between the loading markers 55 and 72 kDa. I am not sure which band is NFE2L2. Do you have data concerning NFE2L2 expression in mouse tissues? According to the literature, it is supposed to be highly expressed in the kidney, the liver and the lung but I do not see a clear expected band in the lung tissue. What is the best way to be sure which band is the good one? Knocking down the gene in a cell line and load the extract on the SDS PAGE gel?
Asked on May 21 2013
Knocking down the gene in a cell line is a good way to determine specificity. I would also recommend adjusting antibody concentration and blocking conditions, prehaps use of BSA instead of milk, to attempt to reduce the non specific binding which you are seeing. If you could provide your detailed protocol I would be happy to review this to see if there may be other optimizations steps which may improve these results.
Answered on May 21 2013