PathScan® RP KEAP1 Sandwich ELISA Kit #99089
Supporting Data
| REACTIVITY | H |
Application Key:
- ELISA-ELISA
Species Cross-Reactivity Key:
- H-Human
Product Information
Product Description
The rapid protocol (RP) PathScan® RP KEAP1 Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of KEAP1 protein in a reduced assay time of 1.5 hours. Incubation of cell lysates and detection antibody on the coated microwell plate forms a sandwich with KEAP1 protein in a single step. The plate is then extensively washed and TMB reagent is added for signal development. The magnitude of absorbance for the developed color is proportional to the quantity of KEAP1 protein. Learn more about your ELISA kit options here.
*Antibodies in this kit are custom formulations specific to kit.
Protocol
Specificity / Sensitivity
Species Reactivity:
Background
The nuclear factor-like 2 (NRF2) transcriptional activator binds antioxidant response elements (ARE) of target gene promoter regions to regulate expression of oxidative stress response genes. Under basal conditions, the NRF2 inhibitor INrf2 (also called KEAP1) binds and retains NRF2 in the cytoplasm where it can be targeted for ubiquitin-mediated degradation (1). Small amounts of constitutive nuclear NRF2 maintain cellular homeostasis through regulation of basal expression of antioxidant response genes. Following oxidative or electrophilic stress, KEAP1 releases NRF2, thereby allowing the activator to translocate to the nucleus and bind to ARE-containing genes (2). The coordinated action of NRF2 and other transcription factors mediates the response to oxidative stress (3). Altered expression of NRF2 is associated with chronic obstructive pulmonary disease (COPD) (4). NRF2 activity in lung cancer cell lines directly correlates with cell proliferation rates, and inhibition of NRF2 expression by siRNA enhances anti-cancer drug-induced apoptosis (5).
KEAP1 contains an amino terminal BTB/POZ domain and a carboxyl terminal KELCH domain (6,7). The KELCH domain is required for interaction with NRF2, and the BTB/POZ domain functions in binding Cul3 E3 ubiquitin ligase (8-10). Under normal conditions, the complex leads to the cytoplasmic sequestration and ubiquitin-mediated proteasomal degradation of NRF2. Electrophilic modification of KEAP1 leads to disassociation of the NRF2/KEAP1 complex. KEAP1 also targets the down regulation of NF-κB activity by targeting IKKβ degradation (11). Mutation of the corresponding KEAP1 gene is seen in lung cancer cases and can lead to uncontrolled activation of NRF2 (12-14).
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- Nguyen, T. et al. (2005) J Biol Chem 280, 32485-92.
- Jaiswal, A.K. (2004) Free Radic Biol Med 36, 1199-207.
- Suzuki, M. et al. (2008) Am J Respir Cell Mol Biol 39, 673-82.
- Homma, S. et al. (2009) Clin Cancer Res 15, 3423-32.
- Itoh, K. et al. (1999) Genes Dev 13, 76-86.
- Dhakshinamoorthy, S. and Jaiswal, A.K. (2001) Oncogene 20, 3906-17.
- Furukawa, M. and Xiong, Y. (2005) Mol Cell Biol 25, 162-71.
- Zhang, D.D. et al. (2004) Mol Cell Biol 24, 10941-53.
- Kobayashi, A. et al. (2004) Mol Cell Biol 24, 7130-9.
- Lee, D.F. et al. (2009) Mol Cell 36, 131-40.
- Padmanabhan, B. et al. (2006) Mol Cell 21, 689-700.
- Singh, A. et al. (2006) PLoS Med 3, e420.
- Ohta, T. et al. (2008) Cancer Res 68, 1303-9.
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