Nuclear factor, erythroid 2 like protein 2 (NFE2L2) commonly referred to as
Nrf2 is an important protein in the regulation of oxidative and xenobiotic
stress response. Kelch-like ECH-associated protein 1 (KEAP1), also known
as inhibitor of Nrf2 (iNrf2), is equally important due to it being the primary
regulator of Nrf2.
In the cytosol, KEAP1 normally binds to Nrf2 preventing
it from translocating to the nucleus and targeting it for ubiquitination and
subsequent degradation by proteasomes. However, KEAP1 is a cysteine
rich protein that is sensitive to oxidative stress. When exposed to the
appropriate stressor, oxidation of C151, C273 and C288 can produce a
conformation change in Keap1 causing release of Nrf2 which then allows
translocation of Nrf2 to the nucleus. Inside the nucleus, Nrf2 is free to
participate in Nrf2/sMAF/ARE transcription pathways and is capable of
activating more than three dozen xenobiotic and oxidative stress response
genes. Many studies have shown a positive correlation between active
Nrf2 and disease amelioration whereas others have shown heightened
NRF2 and/or lower KEAP1 levels to increase cancer cell viability and
resistance to chemotherapy.
In either role KEAP1 regulation of Nrf2 has
emerged as a very important target for researchers trying to ascertain the
full potential of the KEAP1/Nrf2/ARE pathway in terms of lessening the
Deleterious effects of disease and aging.
The NWLSS™ Human KEAP1 ELISA is a sandwich format Enzyme-Linked
Immunosorbent Assay (ELISA). The assay features a microtiter plate pre
coated with a monoclonal antibody specific to human or mouse KEAP1. Briefly, Nrf2
in samples and standards is captured by the plate bound antibody and
remaining liquid is removed. Next, a biotinylated antibody with specificity
to KEAP1 is added and allowed to bind to KEAP1 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 KEAP1 is present. Finally, a sulfuric acid stop solution is added and the resulting yellow colored product is
measured at 450nm. The amount of KEAP1 in the sample can be easily
determined by direct comparison of unknown sample absorbance with
known standard curve absorbance values generated in the assay.