Human Phospho-Histone H2AX (S139) 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 phosphorylated human Histone H2AX (S139) in cell lysates. An immobilized capture antibody specific for Histone H2AX binds both phosphorylated and unphosphorylated Histone H2AX. After washing away unbound material, a biotinylated detection antibody is used to detect only phosphorylated receptor, utilizing a standard HRP format.
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- 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
The Human Phospho-Axl DuoSet IC ELISA is more sensitive than immunoprecipitation (IP)-Western blot analysis. A172 human glioblastoma cells were treated with Gas-6-containing conditioned media1 from the human lung cell line, WI38, for fifteen minutes to induce tyrosine phosphorylation of Axl. Lysates were serially diluted and analyzed by (A) IP-Western blot and (B) this DuoSet IC ELISA. IPs were done using an Anti-Human Axl Monoclonal Antibody and Anti-Mouse IgG Agarose. Immunoblots were incubated with a Biotinylated Anti-Human Phospho-tyrosine Monoclonal Antibody (Catalog # BAM1676) to detect phospho-Axl. Bands were visualized with Streptavidin-HRP (Catalog # DY998) followed by chemiluminescent detection.
The Human Phospho-Axl DuoSet IC ELISA detects ligand-induced Axl tyrosine phosphorylation. A172 cells were untreated or treated with Gas-6-containing conditioned media1 from the human lung cell line, WI38, for 15 minutes. ELISA and IP-Western blot (inset) analyses were done using 100 µg and 400 µg of lysate, respectively. IP-Western blots for phospho-Axl (p-Axl) were done as described in Figure 1. Blots were stripped and total Axl (Axl) was detected using a Biotinylated Anti-Human Axl Polyclonal Antibody (Catalog # BAF154).
The specificity of the Human Phospho-Axl DuoSet IC ELISA is confirmed by receptor competition. A172 cells were treated with Gas-6-containing conditioned media1 from the human lung cell line, WI38, for 15 minutes. The indicated amounts of recombinant extracellular domains of human Axl/Fc Chimera (Catalog # 154-AL), human Dtk/Fc Chimera (Catalog # 859-DK) or human TrkA/Fc Chimera (Catalog # 175-TK) were added to 125 µg of lysate and analyzed using this DuoSet IC ELISA. Competition was observed only with recombinant human Axl.
Preparation and Storage
Background: Histone H2AX
Histone H2AX is one of a number of core histone proteins. In the cellular response to genotoxic insults, ATM and related protein kinases phosphorylate the carboxyl-terminal tail of the H2AX protein (gamma-H2AX). gamma-H2AX marks the site of damage and provides a nucleation site for the formation of damage response and repair complexes. BRCA1 carboxyl terminal (BRCT) domain-containing proteins are recruited to gamma-H2AX to create a protein scaffold for further assembly of signaling complexes.
Citations for Human Phospho-Histone H2AX (S139) 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.
Citations: Showing 1 - 2
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Decreased NHE3 expression in colon cancer is associated with DNA damage, increased inflammation and tumor growth
Authors: D Laubitz, MA Gurney, M Midura-Kie, C Clutter, DG Besselsen, H Chen, FK Ghishan, PR Kiela
Scientific Reports, 2022;12(1):14725.
Sample Types: Cell Lysates
Disturbance of cellular homeostasis as a molecular risk evaluation of human endothelial cells exposed to nanoparticles
Authors: P Wigner, K Zielinski, S Michlewska, P Danielska, A Marczak, EJ Ricci, R Santos-Oli, M Szwed
Scientific Reports, 2021;11(1):3849.
Sample Types: Cell Lysates
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