Transcription factor Nrf2: examination of nuclear protein levels by immunoblotting and promoter response element binding by chromatin immunoprecipitation (ChIP).

Nuclear factor erythroid 2 (NF-E2) related factor 2 (Nrf2) is a transcription factor that governs the expression of over a hundred so-called phase II detoxification and antioxidant genes that are regulated through the antioxidant response element (ARE). Loss of Nrf2 activity has been implicated in cardiovascular disease, inflammation, aging, and cancer. Nrf2 is induced to accumulate in the nucleus when the cell encounters an oxidative stress, a fact that has been exploited experimentally to test the conditions under which ARE-containing genes are expressed. The nuclear levels of Nrf2 give an indication of whether an experimental treatment results in Nrf2 localization and induction. mRNA levels of phase II genes may be measured as a follow-up, but in order to show a direct link between nuclear Nrf2 accumulation and increases in gene expression, it is useful to show that Nrf2 binds to AREs in the promoters of target genes. The simplest way to do this is to employ a chromatin immunoprecipitation (ChIP) assay along with an examination of cellular Nrf2 levels by immunoblotting.