Overview

  • Product name
    Anti-Histone H3 (acetyl K9) antibody - ChIP Grade
    See all Histone H3 primary antibodies
  • Description
    Rabbit polyclonal to Histone H3 (acetyl K9) - ChIP Grade
  • Host species
    Rabbit
  • Specificity
    Specific for acetyl K9, but cross-reacts slightly with K27.
  • Tested applications
    Suitable for: IHC-Fr, IHC-P, ChIP, IP, WB, CHIPseq, ICC/IF, Flow Cytmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Cow, Human, Saccharomyces cerevisiae, Arabidopsis thaliana, Caenorhabditis elegans, Drosophila melanogaster, Zebrafish, Common marmoset, Rice
    Predicted to work with: a wide range of other species
  • Immunogen

    Synthetic peptide corresponding to Human Histone H3 aa 1-100 (N terminal) (acetyl K9) conjugated to keyhole limpet haemocyanin.
    (Peptide available as ab16635)

  • Positive control
    • ChIP: HeLa whole cell extract. WB: Calf thymus histone preparation. Rat dorsal spinal cord tissue lysate. ICC/IF: HeLa cells. Scriptaid treated A549 cells. Arabidopsis thaliana root tips. IHC-P: Human breast carcinoma tissue. IHC-Fr: Mouse thyroid tissue.

Properties

Applications

Our Abpromise guarantee covers the use of ab10812 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
IHC-Fr 1/500.
IHC-P Use a concentration of 1 µg/ml. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
ChIP Use 2-4 µg for 25 µg of chromatin.
IP Use at 20 µg/mg of lysate.
WB 1/500. Detects a band of approximately 17 kDa (predicted molecular weight: 15 kDa).
CHIPseq Use at an assay dependent concentration. PubMed: 22196736
ICC/IF Use a concentration of 1 µg/ml.
Flow Cyt Use at an assay dependent concentration.

ab171870 - Rabbit polyclonal IgG, is suitable for use as an isotype control with this antibody.

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 H3 family.
  • Developmental stage
    Expressed during S phase, then expression strongly decreases as cell division slows down during the process of differentiation.
  • Post-translational
    modifications
    Acetylation is generally linked to gene activation. Acetylation on Lys-10 (H3K9ac) impairs methylation at Arg-9 (H3R8me2s). Acetylation on Lys-19 (H3K18ac) and Lys-24 (H3K24ac) favors methylation at Arg-18 (H3R17me).
    Citrullination at Arg-9 (H3R8ci) and/or Arg-18 (H3R17ci) by PADI4 impairs methylation and represses transcription.
    Asymmetric dimethylation at Arg-18 (H3R17me2a) by CARM1 is linked to gene activation. Symmetric dimethylation at Arg-9 (H3R8me2s) by PRMT5 is linked to gene repression. Asymmetric dimethylation at Arg-3 (H3R2me2a) by PRMT6 is linked to gene repression and is mutually exclusive with H3 Lys-5 methylation (H3K4me2 and H3K4me3). H3R2me2a is present at the 3' of genes regardless of their transcription state and is enriched on inactive promoters, while it is absent on active promoters.
    Methylation at Lys-5 (H3K4me), Lys-37 (H3K36me) and Lys-80 (H3K79me) are linked to gene activation. Methylation at Lys-5 (H3K4me) facilitates subsequent acetylation of H3 and H4. Methylation at Lys-80 (H3K79me) is associated with DNA double-strand break (DSB) responses and is a specific target for TP53BP1. Methylation at Lys-10 (H3K9me) and Lys-28 (H3K27me) are linked to gene repression. Methylation at Lys-10 (H3K9me) is a specific target for HP1 proteins (CBX1, CBX3 and CBX5) and prevents subsequent phosphorylation at Ser-11 (H3S10ph) and acetylation of H3 and H4. Methylation at Lys-5 (H3K4me) and Lys-80 (H3K79me) require preliminary monoubiquitination of H2B at 'Lys-120'. Methylation at Lys-10 (H3K9me) and Lys-28 (H3K27me) are enriched in inactive X chromosome chromatin.
    Phosphorylated at Thr-4 (H3T3ph) by GSG2/haspin during prophase and dephosphorylated during anaphase. Phosphorylation at Ser-11 (H3S10ph) by AURKB is crucial for chromosome condensation and cell-cycle progression during mitosis and meiosis. In addition phosphorylation at Ser-11 (H3S10ph) by RPS6KA4 and RPS6KA5 is important during interphase because it enables the transcription of genes following external stimulation, like mitogens, stress, growth factors or UV irradiation and result in the activation of genes, such as c-fos and c-jun. Phosphorylation at Ser-11 (H3S10ph), which is linked to gene activation, prevents methylation at Lys-10 (H3K9me) but facilitates acetylation of H3 and H4. Phosphorylation at Ser-11 (H3S10ph) by AURKB mediates the dissociation of HP1 proteins (CBX1, CBX3 and CBX5) from heterochromatin. Phosphorylation at Ser-11 (H3S10ph) is also an essential regulatory mechanism for neoplastic cell transformation. Phosphorylated at Ser-29 (H3S28ph) by MLTK isoform 1, RPS6KA5 or AURKB during mitosis or upon ultraviolet B irradiation. Phosphorylation at Thr-7 (H3T6ph) by PRKCBB is a specific tag for epigenetic transcriptional activation that prevents demethylation of Lys-5 (H3K4me) by LSD1/KDM1A. At centromeres, specifically phosphorylated at Thr-12 (H3T11ph) from prophase to early anaphase, by DAPK3 and PKN1. Phosphorylation at Thr-12 (H3T11ph) by PKN1 is a specific tag for epigenetic transcriptional activation that promotes demethylation of Lys-10 (H3K9me) by KDM4C/JMJD2C. Phosphorylation at Tyr-42 (H3Y41ph) by JAK2 promotes exclusion of CBX5 (HP1 alpha) from chromatin.
    Monoubiquitinated by RAG1 in lymphoid cells, monoubiquitination is required for V(D)J recombination (By similarity). Ubiquitinated by the CUL4-DDB-RBX1 complex in response to ultraviolet irradiation. This may weaken the interaction between histones and DNA and facilitate DNA accessibility to repair proteins.
  • Cellular localization
    Nucleus. Chromosome.
  • Information by UniProt
  • Database links
  • Alternative names
    • H3 histone family, member A antibody
    • H3/A antibody
    • H31_HUMAN antibody
    • H3FA antibody
    • Hist1h3a antibody
    • HIST1H3B antibody
    • HIST1H3C antibody
    • HIST1H3D antibody
    • HIST1H3E antibody
    • HIST1H3F antibody
    • HIST1H3G antibody
    • HIST1H3H antibody
    • HIST1H3I antibody
    • HIST1H3J antibody
    • histone 1, H3a antibody
    • Histone cluster 1, H3a antibody
    • Histone H3.1 antibody
    • Histone H3/a antibody
    • Histone H3/b antibody
    • Histone H3/c antibody
    • Histone H3/d antibody
    • Histone H3/f antibody
    • Histone H3/h antibody
    • Histone H3/i antibody
    • Histone H3/j antibody
    • Histone H3/k antibody
    • Histone H3/l antibody
    see all

Images

  • 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 25µg of chromatin, 2µg of  ab10812 (blue), and 20µl 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). Primers and probes are located in the first kb of the transcribed region.
        

  • All lanes : Anti-Histone H3 (acetyl K9) antibody - ChIP Grade (ab10812) at 1 µg/ml

    Lane 1 : Calf Thymus Histone Preparation Nuclear Lysate
    Lane 2 : Calf Thymus Histone Preparation Nuclear Lysate with Human Histone H3 (unmodified ) peptide (ab2903) at 0.5 µg/ml
    Lane 3 : Calf Thymus Histone Preparation Nuclear Lysate with Human Histone H3 (acetyl K9) peptide (ab16635) at 0.5 µg/ml
    Lane 4 : Calf Thymus Histone Preparation Nuclear Lysate with Histone H3 peptide - acetyl K14 at 0.5 µg/ml
    Lane 5 : Calf Thymus Histone Preparation Nuclear Lysate with Human Histone H3 (acetyl K18) peptide (ab24003) at 0.5 µg/ml
    Lane 6 : Calf Thymus Histone Preparation Nuclear Lysate with Human Histone H3 (acetyl K27) peptide (ab24404) at 0.5 µg/ml
    Lane 7 : Calf Thymus Histone Preparation Nuclear Lysate with Human Histone H3 (acetyl K23) peptide (ab48359) at 0.5 µg/ml

    Lysates/proteins at 0.5 µg per lane.

    Secondary
    All lanes : Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/5000 dilution

    Developed using the ECL technique.

    Performed under reducing conditions.

    Predicted band size: 15 kDa
    Observed band size: 17 kDa
    why is the actual band size different from the predicted?


    Exposure time: 30 seconds
  • ICC/IF image of ab10812 stained HeLa cells. The cells were 100% Methanol fixed (5 min) and then incubated in 1%BSA / 10% normal Goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab10812, 1µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 Goat anti-Rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM. This antibody also gave a positive result in 100% Methanol fixed (5 min) Hek293, HepG2, and MCF-7 cells at 5µg/ml.
  • IHC image of Histone H3 (acetyl K9) staining in human breast carcinoma FFPE section, performed on a BondTM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab10812, 1µg/ml, for 8 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.
  • All lanes : Anti-Histone H3 (acetyl K9) antibody - ChIP Grade (ab10812) at 1/1000 dilution

    Lanes 1-2 : Tissue lysate prepared from rat dorsal spinal cord at 5 µg
    Lanes 3-4 : Tissue lysate prepared from rat dorsal spinal cord at 8 µg
    Lane 5 : Tissue lysate prepared from rat dorsal spinal cord at 8 µg with Human Histone H3 (acetyl K9) peptide (ab16635)

    Secondary
    All lanes : HRP conjugated rabbit polyclonal

    Developed using the ECL technique.

    Predicted band size: 15 kDa
    Observed band size: 17 kDa why is the actual band size different from the predicted?


    Exposure time: 2 minutes


    Each sample is from a different extract.

    See Abreview

  • ab10812 staining Histone H3 (acetyl K9) in Mouse thyroid tissue sections by Immunohistochemistry (IHC-Fr - frozen sections). Tissue was fixed with paraformaldehyde, permeabilized with 0.1% Tween 20 and blocked with 10% serum for 30 minutes at 24°C. Samples were incubated with primary antibody (1/200 in 10% serum in PBS) for 1 hour at 24°C. An Alexa Fluor® 555-conjugated Goat anti-rabbit IgG polyclonal (1/500) was used as the secondary antibody.

    See Abreview

  • ab10812 staining histone H3 (acetyl K9) in A549 cells treated with scriptaid (ab120883), by ICC/IF. Increase in histone H3 (acetyl K9) expression correlates with increased concentration of scriptaid, as described in literature.
    The cells were incubated at 37°C for 24 hour in media containing different concentrations of ab120883 (scriptaid) in DMSO, fixed with 4% formaldehyde for 10 minutes at room temperature and blocked with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% tween for 2h at room temperature. Staining of the treated cells with ab10812 (0.1 μg/ml) was performed overnight at 4°C in PBS containing 1% BSA and 0.1% tween. A anti-rabbit DyLight 488 secondary antibody (ab96899) at 1/250 dilution was used as the secondary antibody. Nuclei were counterstained with DAPI and are shown in blue.

  • ab10812 staining Histone H3 (acetyl K9) in the root tips of Arabidopsis thaliana by Immunocytochemistry/ Immunofluorescence. Cells were fixed in 2% paraformaldehyde for 30 minutes at room temperature. Blocking and permeabilization was carried with 4% BSA solution containing 0,5% Triton X-100 in PBS at room temperature for 45 minutes. Slides were washed in PBS and incubated with primary antibody at a 1/200 dilution in 1% PBS for 1 hour at 37°C. Slides were washed in PBS and incubated with the secondary antibody ab6639 (Goat anti-rabbit Cy3 ® (H&L)) at a 1/500 dilution in 1% BSA in PBS for 1 hour at 37°C. Slides were counterstained with DAPI (2µg/mL) for 10 minutes at room temperature.Left image: DAPI stained interphase nucleus with prominent chromocentersMiddle image: Distribution of Histone H3 (acetyl K9) in the nucleus (arrows indicate absence of signal from chromocenters)Right image: Merged image

    See Abreview

References

This product has been referenced in:
  • Jing H  et al. Epigenetic inhibition of Wnt pathway suppresses osteogenic differentiation of BMSCs during osteoporosis. Cell Death Dis 9:176 (2018). WB, ChIP ; Mouse . Read more (PubMed: 29416009) »
  • Hashemi P  et al. Compounds producing an effective combinatorial regimen for disruption of HIV-1 latency. EMBO Mol Med 10:160-174 (2018). WB ; Human . Read more (PubMed: 29246970) »
See all 144 Publications for this product

Customer reviews and Q&As

1-5 of 5 Abreviews or Q&A

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
ChIP
Sample
Mouse Tissue lysate - whole (spinal cord)
Specification
spinal cord
Type
Cross-linking (X-ChIP)
Duration of cross-linking step: 10 minute(s) and 0 second(s)
Specification of the cross-linking agent: formaldehyde
Detection step
Real-time PCR
Positive control
total H3 content (ab1791 antibody), 10% input (not shown here)
Negative control
gene desert primer pair, igg (not shown, but very low values)

Abcam user community

Verified customer

Submitted Jan 06 2012

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
ChIP
Sample
Saccharomyces cerevisiae Cell lysate - nuclear (S288c)
Specification
S288c
Type
Cross-linking (X-ChIP)
Duration of cross-linking step: 15 minute(s) and 0 second(s)
Specification of the cross-linking agent: 1% Fromaldehyde
Detection step
Real-time PCR

Abcam user community

Verified customer

Submitted Aug 02 2011

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
ChIP
Sample
Saccharomyces cerevisiae Cell lysate - nuclear (W303 background)
Specification
W303 background
Type
Cross-linking (X-ChIP)
Duration of cross-linking step: 20 minute(s) and 0 second(s)
Specification of the cross-linking agent: 1% Formaldehyde
Detection step
Semiquantitative PCR

Abcam user community

Verified customer

Submitted May 31 2011

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
ChIP
Sample
Human Cell lysate - nuclear (HeLa)
Specification
HeLa
Type
Cross-linking (X-ChIP)
Duration of cross-linking step: 10 minute(s) and 0 second(s)
Specification of the cross-linking agent: 1% formaldehyde
Detection step
Real-time PCR
Positive control
GAPDH promoter
rabbit IgG (signal <0.2% of input, data not shown)
Negative control
bglo promoter

Abcam user community

Verified customer

Submitted Jul 26 2010

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
ChIP
Sample
Human Cell lysate - nuclear (Jurkat cells)
Specification
Jurkat cells
Type
Cross-linking (X-ChIP)
Duration of cross-linking step: 10 minute(s) and 0 second(s)
Specification of the cross-linking agent: 1% formaldehyde
Detection step
Real-time PCR
Positive control
Total Histone 3 antibody from Upstate
Negative control
IgG

Abcam user community

Verified customer

Submitted May 26 2010

Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"

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