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Figure 1. Phospho-H2A.X (Ser139) Dual Detect CELISA. HeLa cells were seeded at 1x104 cells per well, cultured for 24 hr, serum starved 16 hr, and st...
H2A.X (Ser139) Dual Detect CELISA ASSAY Kit (Fluorogenic Detection)
Description:
H2A.X (Ser139) Dual Detect CELISA ASSAY Kit (Fluorogenic Detection)
Trade Name:
Dual Detect CELISA
Qty/Pk:
96 Assays
Product Overview:
Dual-Detect CELISA is a simple and efficient cell-based ELISA assay to measure the relative expression levels of both the total and the phosphorylated form of H2A.X in whole cells in a 96 well microplate format using dual fluorescent detection. This allows for the normalization of total protein between wells, thus allowing a more accurate measurement of phosphorylation levels among different cells and/or growth conditions. Cells are cultured directly in the microplate and stimulated as desired. They are then fixed and simultaneously incubated with two primary antibodies. One is a phosphospecific antibody directed against the phosphorylated epitope that correlates with H2A.X activation and the other is a total antibody that recognizes the H2A.X target protein, regardless of phosphorylation status. Both the total target protein and the phosphorylated protein are measured in a single well using a double immunoenzymatic labeling procedure, including both horseradish-peroxidase (HRP) and alkaline phosphatase (AP), and two spectrally distinct fluorogenic substrates for HRP and AP. The fluorescence of the phosphorylated protein is normalized to the total protein in each well, thus reducing well-to-well variations.
Background Information:
The histone H2A.X protein is a variant member of the H2A family of histones that is distinguished from other H2A histones by a unique carboxy-terminal sequence. This unique sequence is highly conserved throughout eukaryotic evolution [reviewed in 1] and is rapidly phosphorylated by ATM or ATR at the fourth residue from the carboxy-terminus (Serine 139 in mammalian H2A.X) in response to DNA double-strand breaks (DSBs) [2]. Phosphorylation of H2A.X is important in the formation of a stable repair complex at the site of DNA damage.
H2A.X phosphorylation is a very rapid response to DNA damage, occurring within as little as one minute after exposure to ionizing radiation [2]. Phosphorylation of H2A.X occurs irrespective of the cause of the DNA DSBs and phospho-H2A.X has been observed in response to environmental stresses that result in DSBs as well as programmed cellular events, including DNA rearrangement and apoptosis.
H2A.X phosphorylation is a very rapid response to DNA damage, occurring within as little as one minute after exposure to ionizing radiation [2]. Phosphorylation of H2A.X occurs irrespective of the cause of the DNA DSBs and phospho-H2A.X has been observed in response to environmental stresses that result in DSBs as well as programmed cellular events, including DNA rearrangement and apoptosis.
Key Applications:
ELISA
Species Reactivity:
- Human
- Mouse
- Rat
Usage Statement:
Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.
Entrez Gene Summary:
Histones are basic nuclear proteins that are responsible for the nucleosome structure of the chromosomal fiber in eukaryotes. Two molecules of each of the four core histones (H2A, H2B, H3,
and H4) form an octamer, around which approximately 146 bp of DNA is wrapped in repeating units, called nucleosomes. The linker histone, H1, interacts with linker DNA between nucleosomes and functions in the compaction of chromatin into higher order structures. This gene encodes a member of the histone H2A family, and generates two transcripts through the use of the conserved stem-loop termination motif, and the polyA addition motif.
and H4) form an octamer, around which approximately 146 bp of DNA is wrapped in repeating units, called nucleosomes. The linker histone, H1, interacts with linker DNA between nucleosomes and functions in the compaction of chromatin into higher order structures. This gene encodes a member of the histone H2A family, and generates two transcripts through the use of the conserved stem-loop termination motif, and the polyA addition motif.
UniProt Summary:
Variant histone H2A which replaces conventional H2A in a subset of nucleosomes. Nucleosomes wrap and compact DNA into chromatin, limiting DNA accessibility to the cellular machineries which require DNA as a template. Histones thereby play a central role in transcription regulation, DNA repair, DNA replication and chromosomal stability. DNA accessibility is regulated via a complex set of post-translational modifications of histones, also called histone code, and nucleosome remodeling. Required for checkpoint-mediated arrest of cell cycle progression in response to low doses of ionizing radiation and for efficient repair of DNA double strand breaks (DSBs) specifically when modified by C-terminal phosphorylation.
Quality Assurance:
Quality Control Testing: The reagents in this kit have been optimized to reduce background signal while maintaining a high signal to noise ratio. Quality control testing is performed using the enclosed kit components while following the protocol described.
Components:
500X anti-H2A.X (Total) Antibody
500X anti-phospho-H2A.X (Ser139) Antibody
Black Clear Bottom 96-well Cell Culture Plate
20% Tween® 20 (v/v)
20X TBS
10% BSA in TBS (Blocking Buffer)
Substrate Diluent
500X Goat anti-Mouse HRP Detection Reagent
500X Goat anti-Rabbit AP Detection Reagent
200X HRP Substrate:
200X AP Substrate
500X anti-phospho-H2A.X (Ser139) Antibody
Black Clear Bottom 96-well Cell Culture Plate
20% Tween® 20 (v/v)
20X TBS
10% BSA in TBS (Blocking Buffer)
Substrate Diluent
500X Goat anti-Mouse HRP Detection Reagent
500X Goat anti-Rabbit AP Detection Reagent
200X HRP Substrate:
200X AP Substrate
Brand Family:
Upstate
UniProt Number:
Storage Conditions:
Kit components arrive on dry ice, and must immediately be stored at the temperatures specified on the vials.
This kit is shipped at -20°C. Upon receipt, check individual components for storage conditions. The cell culture plate should be stored at room temperature. The 20X TBS, 20% Tween® 20, 10% BSA and Substrate Diluent should be stored at 4°C. All other components are stored at -20°C. Aliquot the -20°C components, if necessary, to avoid freeze-thaw cycles.
Kit components are stable for 3 months from date of shipment when stored as directed.
This kit is shipped at -20°C. Upon receipt, check individual components for storage conditions. The cell culture plate should be stored at room temperature. The 20X TBS, 20% Tween® 20, 10% BSA and Substrate Diluent should be stored at 4°C. All other components are stored at -20°C. Aliquot the -20°C components, if necessary, to avoid freeze-thaw cycles.
Kit components are stable for 3 months from date of shipment when stored as directed.
Detection Methods:
Fluorescent
Modifications:
Phosphorylation
Gene Symbol:
- H2A.X
- H2AFX
- H2A histone family, member X
Product Name:
H2A.X (Ser139) Dual Detect CELISA ASSAY Kit (Fluorogenic Detection)
Entrez Gene Number:
Alternate Names:
H2A.X
H2AFX
H2A histone family, member X
H2AFX
H2A histone family, member X