Human Phospho-Histone H2AX (S139) DuoSet IC ELISA

Catalog # Availability Size / Price Qty
DYC2288E
DYC2288-5
DYC2288-2
Ancillary Products Available
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Product Details
Citations (2)
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Human Phospho-Histone H2AX (S139) DuoSet IC ELISA Summary

Assay Type
Solid Phase Sandwich ELISA
Format
96-well strip plate
Sample Volume Required
Cell lysates (100 µL)
Sufficient Materials
Kits available for two, five, or fifteen 96-well plates*
Specificity
Please see the product datasheet

* 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.

 

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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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.

Product Datasheets

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Preparation and Storage

Shipping
The product is shipped at ambient temperature. Upon receipt, store it immediately at the temperature recommended below.
Stability & Storage
Store the unopened product at 2 - 8 °C. Do not use past expiration date.

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.

Entrez Gene IDs:
3014 (Human)
Alternate Names:
H2A.X; H2A/X; H2AFX; Histone H2AX

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.

2 Citations: Showing 1 - 2
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  1. 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, 2022-08-30;12(1):14725.
    Species: Human
    Sample Types: Cell Lysates
  2. 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, 2021-02-15;11(1):3849.
    Species: Human
    Sample Types: Cell Lysates

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