Anti-Histone H2A.X antibody - ChIP Grade [EPR22820-23] (ab229914)

Rabbit Recombinant Monoclonal H2A.X antibody. Suitable for IP, ChIP, WB, PepArr, IHC-P, ICC/IF, Flow Cyt (Intra) and reacts with Human samples. Cited in 24 publications.

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Images

ChIP - Anti-Histone H2A.X antibody [EPR22820-23] - ChIP Grade (AB229914), expandable thumbnail

Publications

Key facts

Isotype: IgG
Host species: Rabbit
Storage buffer: pH: 7.2 - 7.4
Preservative: 0.01% Sodium azide
Constituents: PBS, 40% Glycerol (glycerin, glycerine), 0.05% BSA
Form: Liquid
Clonality: Monoclonal

Immunogen

The exact immunogen used to generate this antibody is proprietary information.

Reactivity data

Select an application

Product promiseTestedExpectedPredictedNot recommended

	IP	ChIP	WB	PepArr	IHC-P	ICC/IF	Flow Cyt (Intra)
Human	Tested	Tested	Tested	Tested	Tested	Tested	Tested

Tested
Tested

Species	Dilution info	Notes
Species Human	Dilution info 1/30	Notes -

Tested
Tested

Species	Dilution info	Notes
Species Human	Dilution info 5 µg chromatin for 25 µg chromatin	Notes -

Tested
Tested

Species	Dilution info	Notes
Species Human	Dilution info 1/1000	Notes -

Tested
Tested

Species	Dilution info	Notes
Species Human	Dilution info 0.1 µg/mL	Notes -

Tested
Tested

Species	Dilution info	Notes
Species Human	Dilution info 1/200	Notes -

Tested
Tested

Species	Dilution info	Notes
Species Human	Dilution info 1/100	Notes -

Tested
Tested

Species	Dilution info	Notes
Species Human	Dilution info 1/50	Notes -

Associated Products

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Target data

See full target information H2AX

Function

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.

Alternative names

H2AFX, H2AX, Histone H2AX, H2a/x, Histone H2A.X

Recommended products

Rabbit Recombinant Monoclonal H2A.X antibody. Suitable for IP, ChIP, WB, PepArr, IHC-P, ICC/IF, Flow Cyt (Intra) and reacts with Human samples. Cited in 24 publications.

Key facts

Isotype: IgG
Host species: Rabbit
Storage buffer: pH: 7.2 - 7.4
Preservative: 0.01% Sodium azide
Constituents: PBS, 40% Glycerol (glycerin, glycerine), 0.05% BSA
Form: Liquid
Clonality: Monoclonal
Immunogen: The exact immunogen used to generate this antibody is proprietary information.
Clone number: EPR22820-23
Purification technique: Affinity purification Protein A
Concentration

Storage

Shipped at conditions: Blue Ice
Appropriate short-term storage duration: 1-2 weeks
Appropriate short-term storage conditions: +4°C
Appropriate long-term storage conditions: -20°C
Aliquoting information: Upon delivery aliquot
Storage information: Avoid freeze / thaw cycle

Notes

Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb® patents.

This product is a recombinant monoclonal antibody, which offers several advantages including:

- High batch-to-batch consistency and reproducibility
- Improved sensitivity and specificity
- Long-term security of supply
- Animal-free batch production

For more information, read more on recombinant antibodies.

Product promise

We are dedicated to supporting your work with high quality reagents and we are here for you every step of the way should you need us.

In the unlikely event of one of our products not working as expected, you are covered by our product promise.

Full details and terms and conditions can be found here:
Terms & Conditions.

10 product images

ChIP - Anti-Histone H2A.X antibody [EPR22820-23] - ChIP Grade (ab229914)
Chromatin was prepared from HeLa cells according to the Abcam X-ChIP protocol*. Cells were fixed with formaldehyde for 10min.
The ChIP was performed with 25 μg of chromatin, 5 μg of ab229914 (red), or 5 μg of rabbit normal IgG Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730 (gray) and 20μl of Protein A/G sepharose beads. The immunoprecipitated DNA was quantified by real time PCR (Taqman approach for active and inactive loci, Sybr green approach for heterochromatic loci).
Primers and probes are located in the first kb of the transcribed region.
*http://www.abcam.com/resources?keywords=X%20ChIP%20protocol.
Western blot - Anti-Histone H2A.X antibody [EPR22820-23] - ChIP Grade (ab229914)
This blot was developed using a higher sensitivity ECL substrate.
All lanes: Western blot - Anti-Histone H2A.X antibody [EPR22820-23] - ChIP Grade (ab229914) at 1/1000 dilution
Lane 1: Human brain tissue lysate at 20 µg
Lane 2: HeLa (human cervix adenocarcinoma epithelial cell), whole cell lysate at 20 µg
Lane 3: 293T (human embryonic kidney epithelial cell), whole cell lysate at 20 µg
Secondary
Lane 1: VeriBlot for IP secondary antibody (HRP) at 1/1000 dilution
Lanes 2 - 3: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/100000 dilution
Predicted band size: 15 kDa
Observed band size: 16 kDa, 25 kDa
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H2A.X antibody [EPR22820-23] - ChIP Grade (ab229914)
Immunohistochemical analysis of paraffin-embedded Human testis tissue labeling Histone H2A.X with ab229914 at 1/200 dilution (2.56 ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (Rabbit specific IHC polymer detection kit HRP/DAB ab209101). Nuclear staining on the human testis (PMID/ 24059746). The section was incubated with ab229914 for 15 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.
Secondary antibody only control: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (Rabbit specific IHC polymer detection kit HRP/DAB ab209101)
Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.
Immunocytochemistry/ Immunofluorescence - Anti-Histone H2A.X antibody [EPR22820-23] - ChIP Grade (ab229914)
Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized HeLa (human cervix adenocarcinoma epithelial cell) cells labelling Histone H2A.X with ab229914 at 1/100 dilution, followed by ab229914 anti- Histone H2A.X Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary antibody at 1/1000 dilution (Green). Confocal image showing nuclear staining in HeLa cell line is observed. Alexa Fluor® 594 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 dilution (Red). The Nuclear counterstain was DAPI (Blue).
Secondary antibody only control: Secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary at 1/1000 dilution.
Flow Cytometry (Intracellular) - Anti-Histone H2A.X antibody [EPR22820-23] - ChIP Grade (ab229914)
Intracellular flow cytometric analysis of 4% paraformaldehyde fixed, 90% methanol permeabilized HeLa (human cervix adenocarcinoma epithelial cell) cells labelling Histone H2A.X with ab229914 at 1/50 dilution (Red), compared with a Rabbit monoclonal IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) isotype control (Black)and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat anti rabbit IgG (Alexa Fluor^ï¿½ 488, Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077) at 1/2000 dilution was used as the secondary antibody.
Immunoprecipitation - Anti-Histone H2A.X antibody [EPR22820-23] - ChIP Grade (ab229914)
Histone H2A.X was immunoprecipitated from 0.35 mg HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate with ab229914 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab229914 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (VeriBlot for IP Detection Reagent (HRP) ab131366) was used at 1/5000 dilution.
Lane 1: HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate
Lane 2: ab229914 IP in HeLa whole cell lysate
Lane 3: Rabbit monoclonal IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) instead of ab229914 in HeLa whole cell lysate
Blocking and dilution buffer and concentration/ 5% NFDM/TBST.
Exposure time/ 30 seconds.
All lanes: Immunoprecipitation - Anti-Histone H2A.X antibody [EPR22820-23] - ChIP Grade (ab229914)
Predicted band size: 15 kDa
Peptide Array - Anti-Histone H2A.X antibody [EPR22820-23] - ChIP Grade (ab229914)
All batches of ab229914 are tested in Peptide Array against 501 different modified and unmodified histone peptides; each peptide is printed on the array at six concentrations (each in triplicate).
Circle area represents affinity between the antibody and a peptide: all antigen-containing peptides are displayed as red circles, all other peptides as blue circles. The affinity is calculated as area under curve when antibody binding values are plotted against the corresponding peptide concentration. Each circle area is normalized to the peptide with the strongest affinity.
The complete dataset, including full list of all peptides and information on the position of each peptide in the diagram, is available under the Support & downloads section.
Western blot - Anti-Histone H2A.X antibody [EPR22820-23] - ChIP Grade (ab229914)
A 25-kDa band, likely to be ubiquitinated H2A.X, is observed. The molecular weights are consistent with what have been described in the literature (PMID: 24603765)
All lanes: Western blot - Anti-Histone H2A.X antibody [EPR22820-23] - ChIP Grade (ab229914) at 1/1000 dilution
All lanes: HEK-293 (human embryonic kidney epithelial cell), histone extract at 20 µg
Secondary
All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/100000 dilution
Predicted band size: 15 kDa
Observed band size: 16 kDa, 25 kDa
Western blot - Anti-Histone H2A.X antibody [EPR22820-23] - ChIP Grade (ab229914)
ab229914 was shown to specifically react with Histone H2A.X in wild-type HAP1 cells as signal was lost in Histone H2A.X knockout cells. Wild-type and Histone H2A.X knockout samples were subjected to SDS-PAGE. ab229914 and Anti-GAPDH antibody [EPR16891] - Loading Control ab181602 (Rabbit anti-GAPDH loading control) were incubated 1 hour at room temperature at 1/1000 dilution and 1/200,000 dilution respectively. Blots were developed with Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (Goat Anti-Rabbit IgG H&L (HRP) ab97051) secondary antibody at 1/100,000 dilution for 1 hour at room temperature before imaging. The blot was developed on a BIO-RADÂ® ChemiDocâ„¢ MP instrument using the ECL technique. Blocking/Diluting buffer and concentration: 5% NFDM/TBST Exposure Time: 37 seconds
All lanes: Western blot - Anti-Histone H2A.X antibody [EPR22820-23] - ChIP Grade (ab229914) at 1/1000 dilution
Lane 1: Wild-type HAP1 whole cell lysate at 20 µg
Lane 2: Histone H2A.X knockout HAP1 whole cell lysate at 20 µg
Secondary
All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/20000 dilution
Predicted band size: 15 kDa
Observed band size: 16 kDa
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H2A.X antibody [EPR22820-23] - ChIP Grade (ab229914)
Immunohistochemical analysis of paraffin-embedded Human breast carcinoma tissue labeling Histone H2A.X with ab229914 at 1/200 dilution (2.56 ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (Rabbit specific IHC polymer detection kit HRP/DAB ab209101). Nuclear staining on the human breast carcinoma (PMID/ 27006338). The section was incubated with ab229914 for 15 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.
Secondary antibody only control: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (Rabbit specific IHC polymer detection kit HRP/DAB ab209101)
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Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.