Nrf2 is a basic leucine zipper (bZIP) protein functioning to regulate the expression of many antioxidant proteins. Nrf2 can be found in the cytosol where it is bound to the Kelch-like ECH-associated protein 1 (KEAP1)regulatory molecule. In this sequestered condition Nrf2 cannot cross into the nucleus. However, under conditions of oxidative stress, KEAP1 can dissociate from Nrf2 allowing NRf2 to translocate into the nucleus. Inside the nucleus Nrf2 binds to small Maf (sMAF) proteins forming an Nrf2/sMaf heterodimer capable of binding to the Antioxidant Response Element (ARE) on various stress related gene targets.
In recent years, Nrf2/KEAP1 regulation of the Nrf2/sMAF/ARE transcription pathway has been investigated extensively to assess its possible role in aging and disease. Many studies are showing a positive correlation between Nrf2 nuclear translocation and amelioration of disease resulting in increased interest in Nrf2 promotors and KEAP1 agonists as possible therapeutic agents. Other studies show paradoxical outcomes such as when considering Nrf2 in relation to cancer. In that case, it appears that some tumor cells are capable of exploiting the Nrf2 system to upregulate antioxidant expression which seems to confer chemo-protective properties making the tumor more virulent and resistant to therapy.
In either role Nrf2 has emerged as a very important target for researchers attempting to better understand the positive and negative effects of redox related stress response.
The NWLSS™ Nrf2 (NFE2L2) ELISA is a sandwich format Enzyme-Linked Immunosorbent Assay (ELISA). The assay features a microtiter plate pre-coated with a monoclonal antibody specific to either human, mouse, or rat Nrf2.
Briefly, Nrf2 in samples and standards is captured by the plate bound antibody and remaining liquid is removed. Next, a biotinylated antibody with Nrf2 specificity is added and allowed to bind to Nrf2 captured in the previous step. After incubation, non-bound components are removed by washing. Avidin-HRP conjugate is then added and allowed to incubate. After a subsequent thorough wash TMB-substrate solution is added to each well which produces blue coloration wherever Nrf2 is present. Finally, a sulfuric acid stop solution is added and the resulting yellow colored product is measured at 450nm.
The amount of Nrf2 in the sample can be easily determined by direct comparison of unknown sample absorbance with known standard curve absorbance values generated in the assay.