Revision 1
#99089
Store at +4C
PathScan® RP KEAP1 Sandwich ELISA Kit
1 Kit
(96 assays)
Species Cross Reactivity:
H
UniProt ID:
#Q14145
Entrez-Gene Id:
#9817
877-616-CELL (2355)
877-678-TECH (8324)
3 Trask Lane | Danvers | Massachusetts | 01923 | USA
For Research Use Only. Not for Use in Diagnostic Procedures.
| Product Includes | Product # | Quantity | Color | Storage Temp |
|---|---|---|---|---|
| KEAP1 Rabbit mAb Coated Microwells | 30596 | 96 tests | +4C | |
| KEAP1 Rabbit Detection mAb | 63976 | 1 ea | Red (Lyophilized) | +4C |
| HRP Diluent | 13515 | 5.5 ml | Red | +4C |
| TMB Substrate | 7004 | 11 ml | +4C | |
| STOP Solution | 7002 | 11 ml | +4C | |
| Sealing Tape | 54503 | 2 ea | +4C | |
| ELISA Wash Buffer (20X) | 9801 | 25 ml | +4C | |
| Cell Lysis Buffer (10X) | 9803 | 15 ml | -20C |
*The microwell plate is supplied as 12 8-well modules - Each module is designed to break apart for 8 tests.
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.
Specificity/Sensitivity
The PathScan® RP KEAP1 Sandwich ELISA Kit detects endogenous levels of KEAP1 protein. The kit sensitivity is shown in Figure 1. This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.
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).
Background References
- Cullinan, S.B. et al. (2004) Mol Cell Biol 24, 8477-86.
- 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.
Cross-Reactivity Key
H: Human M: Mouse R: Rat Hm: Hamster Mk: Monkey Vir: Virus Mi: Mink C: Chicken Dm: D. melanogaster X: Xenopus Z: Zebrafish B: Bovine Dg: Dog Pg: Pig Sc: S. cerevisiae Ce: C. elegans Hr: Horse GP: Guinea Pig Rab: Rabbit G: Goat All: All Species Expected
Trademarks and Patents
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
PathScan is a registered trademark of Cell Signaling Technology, Inc.
KARPAS cell line source: Dr. Abraham Karpas at the University of Cambridge.
All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.
Limited Uses
Except as otherwise expressly agreed in a writing signed by a legally authorized representative of CST, the following terms apply to Products provided by CST, its affiliates or its distributors. Any Customer's terms and conditions that are in addition to, or different from, those contained herein, unless separately accepted in writing by a legally authorized representative of CST, are rejected and are of no force or effect.
Products are labeled with For Research Use Only or a similar labeling statement and have not been approved, cleared, or licensed by the FDA or other regulatory foreign or domestic entity, for any purpose. Customer shall not use any Product for any diagnostic or therapeutic purpose, or otherwise in any manner that conflicts with its labeling statement. Products sold or licensed by CST are provided for Customer as the end-user and solely for research and development uses. Any use of Product for diagnostic, prophylactic or therapeutic purposes, or any purchase of Product for resale (alone or as a component) or other commercial purpose, requires a separate license from CST. Customer shall (a) not sell, license, loan, donate or otherwise transfer or make available any Product to any third party, whether alone or in combination with other materials, or use the Products to manufacture any commercial products, (b) not copy, modify, reverse engineer, decompile, disassemble or otherwise attempt to discover the underlying structure or technology of the Products, or use the Products for the purpose of developing any products or services that would compete with CST products or services, (c) not alter or remove from the Products any trademarks, trade names, logos, patent or copyright notices or markings, (d) use the Products solely in accordance with CST Product Terms of Sale and any applicable documentation, and (e) comply with any license, terms of service or similar agreement with respect to any third party products or services used by Customer in connection with the Products.
Revision 1
Figure 1. KEAP1 protein is expressed at differing levels across several cell lines. The absorbance at 450 nm for a panel of cell lines using the PathScan® RP KEAP1 Sandwich ELISA Kit #99089 is shown in the upper figure. The corresponding western blot using a KEAP1 antibody is shown in the lower figure. Unstarved cells were harvested and then lysed.
Figure 2. KEAP1 protein is expressed in KARPAS 299 cells. The relationship between lysate protein concentration from KARPAS 299 cells and the absorbance at 450 nm using the PathScan® RP KEAP1 Sandwich ELISA Kit #99089 is shown.
