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.
Human Phospho-Histone H2AX (S139) DuoSet IC ELISA
R&D Systems | Catalog # DYC2288-2
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Key Product Details
Assay Type
Solid Phase Sandwich ELISA
Sample Type
Cell Lysates
Reactivity
Human
Human Phospho-Histone H2AX (S139) DuoSet IC ELISA 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
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Product Summary for Human Phospho-Histone H2AX (S139) DuoSet IC ELISA
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 Specifications
Assay Format
96-well strip plate (sold separately)
Sample Volume Required
Cell lysates (100 µL)
Detection Method
Colorimetric ELISA - 450nm (TMB)
Conjugate
Biotin
Specificity
Label
HRP
Scientific Data Images for Human Phospho-Histone H2AX (S139) DuoSet IC ELISA
Standard Curve
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.Kit Contents for Human Phospho-Histone H2AX (S139) DuoSet IC ELISA
- 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
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
Additional Histone H2AX Products
Product Documents for Human Phospho-Histone H2AX (S139) DuoSet IC ELISA
Certificate of Analysis
To download a Certificate of Analysis, please enter a lot or batch number in the search box below.
Note: Certificate of Analysis not available for kit components.
Product Specific Notices for Human Phospho-Histone H2AX (S139) DuoSet IC ELISA
For research use only
Related Research Areas
Citations for Human Phospho-Histone H2AX (S139) DuoSet IC ELISA
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Protocols
Find general support by application which include: protocols, troubleshooting, illustrated assays, videos and webinars.
- ELISA Sample Preparation & Collection Guide
- ELISA Troubleshooting Guide
- How to Run an R&D Systems DuoSet ELISA
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- Quantikine HS ELISA Kit Assay Principle, Alkaline Phosphatase
- Quantikine HS ELISA Kit Principle, Streptavidin-HRP Polymer
- Sandwich ELISA (Colorimetric) – Biotin/Streptavidin Detection Protocol
- Sandwich ELISA (Colorimetric) – Direct Detection Protocol
- Troubleshooting Guide: ELISA
- View all Protocols, Troubleshooting, Illustrated assays and Webinars
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