Human Total IkB-alpha DuoSet IC ELISA
Human Total IkB-alpha 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.
This DuoSet IC ELISA contains the basic components required for the development of sandwich ELISAs to measure total IkB-alpha in cell lysates. An immobilized capture antibody specific for IkB-alpha binds both phosphorylated and unphosphorylated IkB-alpha. After washing away unbound material, a biotinylated detection antibody is used to detect both phosphorylated and unphosphorylated protein, utilizing a standard Streptavidin-HRP format.
- 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
- Capture Antibody
- Conjugated Detection Antibody
- Calibrated Immunoassay Standard or Control
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
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
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Figure 1: The Human Total IκB-alpha DuoSet IC ELISA specifically recognizes IκB-alpha as shown by Western blot analysis of the protein bound by the capture antibody supplied in the kit. Lysate prepared from MCF-7 human breast cancer cells was incubated in wells coated with Total IκB-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 resolved by SDS-PAGE, transferred to a PVDF membrane, and immunoblotted with Total IκB-alpha Detection Antibody. In addition to IgG heavy and light chains, only a single band at 39 kDa corresponding to IκB-alpha was detected in captured material, indicating that the ELISA is specific for IκB-alpha.
Specificity was further demonstrated using cross-reactivity experiments with related IκB family members. Recombinant human (rh) IκB-beta and rhIκB-epsilon were tested at 50 ng/mL and did not cross-react or interfere in the assay.
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Figure 2: Quantification of total IκB-alpha human cell lysates. Lysates prepared from MCF-7 human breast cancer, Daudi human Burkitt's lymphoma, and HeLa human cervical epithelial carcinoma cells were quantified with this DuoSet® IC ELISA. The same lysates were also immunoblotted (inset) with anti-IκB-alpha monoclonal antibody (Catalog # MAB4299). The DuoSet® IC ELISA results correlate well with the relative amounts of IκB-alpha detected by Western Blot.
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Figure 3: Quantification of total IκB-alpha in TNF-alpha treated HeLa cell lysates. Lysates prepared from HeLa human cervical epithelial carcinoma cells either untreated or treated with 20 ng/mL of recombinant human TNF- alpha (Catalog # 210-TA) for 20 minutes were quantified with this DuoSet® IC ELISA. The same lysates were also immunoblotted (inset) with anti-IκB-beta monoclonal antibody. The DuoSet® IC ELISA results correlate well with the relative amounts of IκB-alpha detected by Western Blot.
Preparation and Storage
The inhibitory IkB proteins (alpha, beta, epsilon) complex with and sequester NFkB family members in the cytoplasm, rendering these transcription factors inactive. When phosphorylated by an active IkB kinase (IKK) complex, IkB proteins are degraded by proteasomes, resulting in the release and nuclear translocation of active NFkB.
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