Overview

  • Product name

    Anti-Histone H2A antibody - ChIP Grade
    See all Histone H2A primary antibodies
  • Description

    Rabbit polyclonal to Histone H2A - ChIP Grade
  • Host species

    Rabbit
  • Tested applications

    Suitable for: ICC/IF, IHC-P, WB, IP, ChIPmore details
  • Species reactivity

    Reacts with: Mouse, Human
  • Immunogen

    Synthetic peptide within Human Histone H2A aa 100 to the C-terminus (C terminal) conjugated to keyhole limpet haemocyanin. The exact sequence is proprietary.
    (Peptide available as ab19751)

  • Positive control

    • WB: HeLa nuclear extract. Calf thymus histone preparation. Histone H2A Recombinant Protein. ChIP: Chromatin from U-2 OS cells. IHC-P: Human testis tissue. IP: Histone H2A IP in HeLa whole cell lysate. ICC/IF: HeLa cells.

Properties

  • Form

    Liquid
  • Storage instructions

    Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C or -80°C. Avoid freeze / thaw cycle.
  • Storage buffer

    Preservative: 0.02% Sodium Azide
    Constituents: 1% BSA, PBS, pH 7.4
  • Concentration information loading...
  • Purity

    Immunogen affinity purified
  • Clonality

    Polyclonal
  • Isotype

    IgG
  • Research areas

Applications

Our Abpromise guarantee covers the use of ab18255 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
ICC/IF 1/200. see Abreview submitted by Kirk McManus
IHC-P 1/150. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
WB Use a concentration of 1 µg/ml. Detects a band of approximately 14 kDa (predicted molecular weight: 14 kDa).
IP Use a concentration of 5 µg/ml.
ChIP Use at an assay dependent concentration. Every new batch of this antibody is tested at Abcam in ChIP

Target

  • Function

    Core component of nucleosome. 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.
  • Sequence similarities

    Belongs to the histone H2A family.
  • Post-translational
    modifications

    The chromatin-associated form is phosphorylated on Thr-121 during mitosis.
    Deiminated on Arg-4 in granulocytes upon calcium entry.
    Monoubiquitination of Lys-120 by RING1 and RNF2/RING2 complex gives a specific tag for epigenetic transcriptional repression and participates in X chromosome inactivation of female mammals. It is involved in the initiation of both imprinted and random X inactivation. Ubiquitinated H2A is enriched in inactive X chromosome chromatin. Ubiquitination of H2A functions downstream of methylation of 'Lys-27' of histone H3. Monoubiquitination of Lys-120 by RNF2/RING2 can also be induced by ultraviolet and may be involved in DNA repair. Following DNA double-strand breaks (DSBs), it is ubiquitinated through 'Lys-63' linkage of ubiquitin moieties by the E2 ligase UBE2N and the E3 ligases RNF8 and RNF168, leading to the recruitment of repair proteins to sites of DNA damage. Monoubiquitination and ionizing radiation-induced 'Lys-63'-linked ubiquitination are distinct events.
    Phosphorylation on Ser-2 is enhanced during mitosis. Phosphorylation on Ser-2 by RPS6KA5/MSK1 directly represses transcription. Acetylation of H3 inhibits Ser-2 phosphorylation by RPS6KA5/MSK1.
    Symmetric dimethylation on Arg-4 by the PRDM1/PRMT5 complex may play a crucial role in the germ-cell lineage.
  • Cellular localization

    Nucleus. Chromosome.
  • Information by UniProt
  • Database links

  • Alternative names

    • H2a 615 antibody
    • H2A antibody
    • H2A GL101 antibody
    • H2A histone family member A antibody
    • H2A.1 antibody
    • H2A.2 antibody
    • H2A/a antibody
    • H2A/m antibody
    • H2A/O antibody
    • H2A/q antibody
    • H2A1B_HUMAN antibody
    • H2AFA antibody
    • H2AFE antibody
    • H2AFL antibody
    • H2AFM antibody
    • H2AFO antibody
    • H2AFQ antibody
    • HIST1H2AE antibody
    • HIST1H2AJ antibody
    • HIST2H2AA antibody
    • HIST2H2AA3 antibody
    • HIST2H2AB antibody
    • HIST2H2AC antibody
    • Histone 1 H2ae antibody
    • Histone 2 H2aa3 antibody
    • Histone 2 H2ab antibody
    • Histone 2 H2ac antibody
    • Histone H2A type 1 B antibody
    • Histone H2A type 1 C antibody
    • Histone H2A type 1 E antibody
    • Histone H2A type 1 J antibody
    • Histone H2A type 1-B/E antibody
    • Histone H2A.2 antibody
    • Histone H2A/a antibody
    • Histone H2A/m antibody
    • MGC74460 antibody
    see all

Images

  • Myc affects the incorporation levels of histone variant H2A.Z.

    qChIP was performed using specific antibodies recognizing A. H2A.Z, B. H2A.Zac, Panel D. H2A, E. H2AK5ac and G. H1. All the qChIP values are expressed as % of input and normalized for total histone H3, with the exception of C and F, where H2A.Z acetylation is noramlized for H2A.Z density, and H2AK5 acetylation is normalized for H2A density, respectively. The box plots show the fold change distribution of each acetylated residue for the two subpopulations.

  • DENV expression disrupts histone oligomerization.

    Panel A and B shown:

    Huh7 cells were transfected with DEN2 C and/or infected with DEN2 24 h post-transfection. Cells were lysed 24 h post-infection (48 h post-transfection) and lysates were run on 4–12% SDS-PAGE gel. Gels were used in a Western blotting assay with antibodies against histones H2A ab18255 (A), H2B (B), H3 (C) and H4 (D); monomers, dimers and octamers are indicated. Gels were stripped and reprobed with an antibody against actin as a protein loading control. The same amount of protein was loaded in each lane for each gel as a control for expression.

  • DENV C colocalizes with histones in Huh7 liver cells.

    DEN2 C colocalized with H2A (A), H2B (B), H3 (C) and H4 (D) in Huh7 cells. Cells were transfected with GFP-DEN2 C and fixed in 4% paraformaldehyde 48 h post-transfection. Cells were stained with antibodies against histones and a TRITC secondary antibody. Cells were counterstained with DAPI to visualize the nucleus. GFP-DEN2 C expression is green, histone staining is red and DAPI is blue.

  • All lanes : Anti-Histone H2A antibody - ChIP Grade (ab18255) at 1 µg/ml

    Lane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate at 10 µg
    Lane 2 : Calf Thymus Histone Preparation Nuclear Lysate at 0.5 µg
    Lane 3 : Histone H2A Recombinant Protein at 0.1 µg
    Lane 4 : Histone H3.1 Recombinant Protein at 0.1 µg
    Lane 5 : Histone H4 Recombinant Protein at 0.1 µg

    Secondary
    All lanes : Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

    Developed using the ECL technique.

    Performed under reducing conditions.

    Predicted band size: 14 kDa
    Observed band size: 17 kDa
    why is the actual band size different from the predicted?


    Exposure time: 3 minutes
  • Chromatin was prepared from U-2 OS (Human bone osteosarcoma epithelial cell line) cells according to the Abcam X-ChIP protocol.

    Cells were fixed with formaldehyde for 10 min. The ChIP was performed with 25 µg of chromatin, 6 µL of ab18255 (blue), and 20 µL of Protein A/G sepharose beads. No antibody was added to the beads control (yellow). 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.

  • Image courtesy of Human Protein Atlas

    ab18255 staining histone H2A in human testis tissue, showing a distinct and strong nuclear staining pattern at cells in ductus seminiferus. Paraffin embedded human tissue was incubated with ab18255 (1/1200 dilution) for 30 mins at room temperature. Antigen retrieval was performed by heat induction in citrate buffer pH 6.
    ab18255 was tested in a tissue microarray (TMA) containing a wide range of normal and cancer tissues as well as a cell microarray consisting of a range of commonly used, well characterised human cell lines. Further results for this antibody can be found at www.proteinatlas.org

    .

  • Histone H2A - ChIP Grade was immunoprecipitated using 0.5 mg HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell extract, 5 µg of Rabbit polyclonal to and 50 µL of protein G magnetic beads (+). No antibody was added to the control (-).

    The antibody was incubated under agitation with Protein G beads for 10 min, HeLa whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10 min under agitation.

    Proteins were eluted by addition of 40 µL SDS loading buffer and incubated for 10 min at 70°C; 10 µL of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab18255.

    Secondary: Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697).

    Band: 14kDa, non specific bands - 42kDa: We are unsure as to the identity of this extra band; Histone H2A - ChIP Grade

  • All lanes : Anti-Histone H2A antibody - ChIP Grade (ab18255) at 1 µg/ml

    Lane 1 : HeLa (Human epithelial cell line from cervix adenocarcinoma) lysate at 20 µg
    Lane 2 : HeLa nuclear lysate at 20 µg
    Lane 3 : Calf thymus histone lysate at 20 µg
    Lane 4 : HeLa lysate at 1 µg/ml with Human Histone H2A peptide (ab19751) at 1 µg/ml
    Lane 5 : HeLa nuclear lysate at 1 µg/ml with Human Histone H2A peptide (ab19751) at 1 µg/ml
    Lane 6 : Calf thymus histone lysate at 1 µg/ml with Human Histone H2A peptide (ab19751) at 1 µg/ml

    Predicted band size: 14 kDa
    Observed band size: 14 kDa
    Additional bands at: 22 kDa (possible cross reactivity)



    ab18255 is partially blocked by the immunizing peptide ab19751. There is an additional band at 22kDa in HeLa lysate which is attributed to cross-reactivity.

  • ICC/IF image of ab18255 stained HeLa (Human epithelial cell line from cervix adenocarcinoma) cells.

    The cells were fixed in 100% methanol (5 min) then permeabilized using 0.1% PBS-Triton and then incubated in 1% BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to further permeabilize the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody ab18255 at 1 µg/ml overnight at +4°C. The secondary antibody (pseudo-colored green) was Alexa Fluor® 488 goat anti- rabbit (ab150081) IgG (H+L) preadsorbed, used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (pseudo-colored red) at a 1/200 dilution for 1h at room temperature. DAPI was used to stain the cell nuclei (pseudo-colored blue) at a concentration of 1.43 µM for 1 hour at room temperature.

  • All lanes : Anti-Histone H2A antibody - ChIP Grade (ab18255) at 1/1000 dilution

    Lane 1 : Native recombinant octamers K562 cells
    Lane 2 : Recombinant Human octamers containing H2A
    Lane 3 : Recombinant Human octamers containing H2A.Z.2.1
    Lane 4 : Recombinant Human octamers containing H2A.Z.1

    Lysates/proteins at 0.5 µg per lane.

    Secondary
    All lanes : HRP-conjugated donkey anti-rabbit IgG polyclonal at 1/10000 dilution

    Developed using the ECL technique.

    Performed under reducing conditions.

    Predicted band size: 14 kDa
    Observed band size: 15 kDa why is the actual band size different from the predicted?


    Exposure time: 5 minutes

    See Abreview

References

This product has been referenced in:

  • Qin B  et al. UFL1 promotes histone H4 ufmylation and ATM activation. Nat Commun 10:1242 (2019). Read more (PubMed: 30886146) »
  • Yuan X  et al. Metformin inhibits glioma cells stemness and epithelial-mesenchymal transition via regulating YAP activity. Biomed Pharmacother 102:263-270 (2018). WB ; Human . Read more (PubMed: 29567539) »
See all 77 Publications for this product

Customer reviews and Q&As

1-10 of 21 Abreviews or Q&A

Application
Western blot
Sample
Mouse Tissue lysate - whole (Brain)
Gel Running Conditions
Reduced Denaturing (4-12% Bis Tris)
Loading amount
25 µg
Specification
Brain
Blocking step
(agent) for 30 minute(s) · Concentration: 0.001% · Temperature: 25°C

Dr. Gillian Hunter

Verified customer

Submitted Feb 15 2019

Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (SAOS)
Permeabilization
Yes - NP40
Specification
SAOS
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: 21°C
Fixative
Formaldehyde

Abcam user community

Verified customer

Submitted Oct 12 2018

Application
Western blot
Sample
Human Cell lysate - nuclear (Vascular smooth muscle cells)
Gel Running Conditions
Reduced Denaturing
Loading amount
30 µg
Treatment
100 uM H2O2
Specification
Vascular smooth muscle cells
Blocking step
Milk as blocking agent for 3 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 21°C

Abcam user community

Verified customer

Submitted Oct 12 2018

Application
Western blot
Sample
Human Cell lysate - whole cell (U2OS)
Gel Running Conditions
Reduced Denaturing (4-20%)
Loading amount
5 µg
Specification
U2OS

Abcam user community

Verified customer

Submitted Oct 05 2016

Application
Western blot
Loading amount
0.5 µg
Gel Running Conditions
Reduced Denaturing (Criterion TGX 4-20% (BioRad))
Sample
Human Purified protein (Recombinant octamers & purified K562 cell histones)
Specification
Recombinant octamers & purified K562 cell histones
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: 20°C

Dr. Ragnhild Eskeland

Verified customer

Submitted Jan 27 2015

Application
Western blot
Loading amount
10000 cells
Gel Running Conditions
Reduced Denaturing (4-12% gradient)
Sample
Mouse Cell lysate - whole cell (MEFs)
Specification
MEFs
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C

Abcam user community

Verified customer

Submitted Dec 04 2014

Application
Western blot
Loading amount
10000 cells
Gel Running Conditions
Reduced Denaturing (4-12% gradient)
Sample
Human Cell lysate - nuclear (Huh7 liver)
Specification
Huh7 liver
Treatment
Infected with dengue virus 24 hours
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C

Abcam user community

Verified customer

Submitted Nov 18 2014

Application
Western blot
Sample
Human Cell lysate - whole cell (Ntera2 cells)
Loading amount
20 µg
Specification
Ntera2 cells
Gel Running Conditions
Reduced Denaturing (15% gel)
Blocking step
Milk as blocking agent for 30 minute(s) · Concentration: 5% · Temperature: 25°C

Abcam user community

Verified customer

Submitted May 29 2012

Answer

ab11175 and ab18255 were never used togteher in Sandwich ELISA. These however can be used in Indirect ELISA.

The sELISA application totally based on difference in epiotopes, the antibodies recognize means the epitopes should not interfere, ab11175 and ab18255 immunogen sequence and hence target refer to two different proteins so these can not be paired in sELISA. You would need two antibodies against the same protein, both of them should be optimized for use as capture or detection.

Please note both of these antibodies are not conjugated so you would need one of them conjugated or you would need a tertiary conjugated antibody. You may experience problems with tertiary antibody because it will detect both capture and detections antibodies because both ab11175 and ab18255 are rabbit; this will give you false results.

I have attached a list of anti H2A and H2A.X antibodies we have in catalogue. Please select the pairs you would like to use. Please consider the following criteria

- immunogen should be different
- species should be different
- One should be conjugated (detection),

Read More
Application
Western blot
Sample
Mouse Cell lysate - nuclear (Mouse embryonic fibroblasts)
Loading amount
30 µg
Specification
Mouse embryonic fibroblasts
Gel Running Conditions
Reduced Denaturing (4-16% Tris-Gly gel)
Blocking step
LI-COR® Odyssey® Blocking Buffer as blocking agent for 45 minute(s) · Concentration: 50% · Temperature: RT°C

Abcam user community

Verified customer

Submitted Feb 23 2012

1-10 of 21 Abreviews or Q&A

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