Human/Rat Total HIF-2 alpha/EPAS1 DuoSet IC ELISA Summary
* Provided that the recommended microplates, buffers, diluents, substrates and solutions are used, and the assay is run as summarized in the Assay Procedure provided.
Product Features
- Optimized capture and detection antibody pairings with recommended concentrations save lengthy development time
- Development protocols are provided to guide further assay optimization
- Assay can be customized to your specific needs
- Available in 2, 5, and 15- (96-well) plate pack sizes
- Economical alternative to Western blot
Kit Content
- Capture Antibody
- Conjugated Detection Antibody
- Calibrated Immunoassay Standard or Control
- Streptavidin-HRP
Other Reagents Required
PBS: (Catalog # DY006), or 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 1.5 mM KH2O4, pH 7.2 - 7.4, 0.2 µm filtered
Wash Buffer: (Catalog # WA126), or equivalent
Lysis Buffer*
IC Diluent*
Blocking Buffer*
Substrate Solution: 1:1 mixture of Color Reagent A (H2O2) and Color Reagent B (Tetramethylbenzidine) (Catalog # DY999)
Stop Solution: 2 N H2SO4 (Catalog # DY994)
Microplates: From Costar EIA Plate (Costar Catalog # 2592) or R&D Systems (Catalog # DY990), or equivalent
Plate Sealers: ELISA Plate Sealers (Catalog # DY992), or equivalent
*For the Lysis Buffer, IC Diluent, and Blocking BUffer recommended for a specific DuoSet ELISA Development Kit, please see the product
Scientific Data
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Total HIF-2 alpha Detection in HepG2 Cell Lysate. Lysate prepared from HepG2 human hepatocellular carcinoma cells treated with 150 μM of CoCl2 for 18 hours were incubated in wells coated with Total HIF-2 alpha Capture Antibody. Unbound material was removed by washing and bound material was solubilized in SDS gel sample buffer. The same lysate and captured proteins were electrophoresed and transferred to a PVDF membrane. The captured protein was immunoblotted with Total HIF-2 alpha Detection Antibody and the lysate was immunoblotted with anti-HIF-2 alpha antibody. Only one band corresponding to HIF-2 alpha was detected in the captured material.Cross-reactivity experiments were performed with this DuoSet IC ELISA to further determine specificity. Recombinant mouse HIF-2 alpha was assayed at 80 ng/mL and read 16.4 ng/mL (20.5% cross-reactivity). Recombinant human (rh) HIF-1 alpha and rhARNT were assayed at 80 ng/mL and did not cross-react or interfere in the assay.
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Total HIF-1 alpha Detection in 786-O, Caki-2, and MCF-7 Cell Lysate. Lysate prepared from 786-O human renal adenocarcinoma cells, Caki-2 human kidney clear carcinoma cells, and MCF-7 human breast cancer cells untreated or treated with 150 μM of CoCl2 for 18 hours were analyzed with both this DuoSet IC ELISA and the Human/Mouse Total HIF-1 alpha DuoSet IC ELISA (R&D Systems, Catalog # DYC1935). The 786-O cells are positive for HIF-2 alpha only while Caki-2 and MCF-7 treated with CoCl2 are positive for HIF-1 alpha only. Only the HIF-2 alpha positive 786-O cells were detected with the Human/Rat Total HIF-2 alpha /EPAS1 DuoSet IC ELISA, indicating that this DuoSet IC ELISA is specific for HIF-2 alpha.
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Total HIF-2 alpha/EPAS1 Detection in 786-O and HepG2 Cell Lysate. Lysates prepared from 786-O human renal adenocarcinoma cell and HepG2 human hepatocellular carcinoma cells untreated or treated with 150 μM of CoCl2 for 18 hours were quantified with this DuoSet IC ELISA. The same lysates were also immunoblotted (inset) with anti-HIF-2 alpha antibody. The DuoSet IC ELISA results correlated well with the relative amounts of HIF-2 alpha detected by Western blot.
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Total HIF-2 alpha/EPAS1 Detection in PC-12 Cell Lysate. Lysates prepared from PC-12 rat adrenal pheochromocytoma cells untreated or treated with 150 μM of CoCl2 for 18 hours were quantified with this DuoSet IC ELISA. The same lysates were also immunoblotted (inset) with anti-HIF-2 alpha antibody (R&D Systems, Catalog # AF2886). The DuoSet IC ELISA results correlate well with the relative amounts of HIF-2 alpha detected by Western blot.
Product Datasheets
Preparation and Storage
Background: HIF-2 alpha/EPAS1
The hypoxia-inducible transcription factor 2 alpha is stabilized in hypoxic tissue and, similarly to HIF-1 alpha, complexes with aryl hydrocarbon receptor nuclear translocator (ARNT). Both the HIF-1 and HIF-2 complexes bind hypoxia-response elements (HREs) in the promoters of many genes involved in adapting to an environment of insufficient oxygen or hypoxia. HIF-1 and HIF-2 do not appear to be completely redundant, although specific functions are only beginning to be elucidated. Hypoxic tissue environments occur in vascular and pulmonary diseases as well as cancer, indicating the potentially broad impact of gene regulation by both HIF-1 alpha and HIF-2 alpha.
Citation for Human/Rat Total HIF-2 alpha/EPAS1 DuoSet IC ELISA
R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.
1 Citation: Showing 1 - 1
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Uncovering pharmacological mechanisms of Wu-tou decoction acting on rheumatoid arthritis through systems approaches: drug-target prediction, network analysis and experimental validation.
Authors: Zhang, Yanqiong, Bai, Ming, Zhang, Bo, Liu, Chunfang, Guo, Qiuyan, Sun, Yanqun, Wang, Danhua, Wang, Chao, Jiang, Yini, Lin, Na, Li, Shao
Sci Rep, 2015-03-30;5(0):9463.
Species: Rat
Sample Types: Serum
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