Overview

  • Product name
    Anti-Histone H3 (acetyl K27) antibody - ChIP Grade
    See all Histone H3 primary antibodies
  • Description
    Rabbit polyclonal to Histone H3 (acetyl K27) - ChIP Grade
  • Host species
    Rabbit
  • Specificity
    All batches of ab4729 are tested using peptide arrays and show less than 30% cross reactivity with both Histone H3 acetyl K9 and unmodified Histone H3 peptides in this application. For further information please see the peptide array image on this datasheet, or contact our technical support team who will be happy to help.
  • Tested applications
    Suitable for: IHC-Fr, ICC/IF, WB, IHC-P, CHIPseq, ChIP/Chip, ChIP, PepArrmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Chicken, Cow, Human, Arabidopsis thaliana, Drosophila melanogaster, Monkey, Zebrafish, Plasmodium falciparum, Rice, Cyanidioschyzon merolae
    Predicted to work with: Xenopus laevis
  • Immunogen

    Synthetic peptide corresponding to Human Histone H3 aa 1-100 (acetyl K27) conjugated to Keyhole Limpet Haemocyanin (KLH).
    (Peptide available as ab56316, ab24404)

  • Positive control
    • ChIP: Chromatin from HeLa cells. IHC-P: Human colon tissue. WB: Butyrate treated HeLa histone nuclear lysate. Calf thymus histone lysate. ICC/IF: Sodium butyrate treated HeLa cells.
  • General notes

    Abcam recommended secondaries - Goat Anti-Rabbit HRP (ab205718) and Goat Anti-Rabbit Alexa Fluor® 488 (ab150077).

    See other anti-rabbit secondary antibodies that can be used with this antibody.

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
    pH: 7.40
    Preservative: 0.02% Sodium azide
    Constituent: PBS

    Batches of this product that have a concentration < 1mg/ml may have BSA added as a stabilising agent. If you would like information about the formulation of a specific lot, please contact our scientific support team who will be happy to help.
  • Concentration information loading...
  • Purity
    Immunogen affinity purified
  • Clonality
    Polyclonal
  • Isotype
    IgG
  • Research areas

Applications

Our Abpromise guarantee covers the use of ab4729 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 Use at an assay dependent concentration.
ICC/IF Use a concentration of 0.5 µg/ml.

Can be used with paraformaldehyde- or methanol- fixed cells.

 

WB Use a concentration of 1 µg/ml. Detects a band of approximately 17 kDa (predicted molecular weight: 15 kDa).

We recommend Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody.

IHC-P Use a concentration of 1 µg/ml. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
CHIPseq Use at an assay dependent concentration.
ChIP/Chip Use at an assay dependent concentration.
ChIP Use 2 µg for 25 µg of chromatin.
PepArr Use a concentration of 0.2 - 0.02 µg/ml.

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 E pseudogene antibody
    • H3 histone family, member A antibody
    • H3/A antibody
    • H31_HUMAN antibody
    • H3F3 antibody
    • H3FA antibody
    • Hist1h3a antibody
    • HIST1H3B antibody
    • HIST1H3C antibody
    • HIST1H3D antibody
    • HIST1H3E antibody
    • HIST1H3F antibody
    • HIST1H3G antibody
    • HIST1H3H antibody
    • HIST1H3I antibody
    • HIST1H3J antibody
    • HIST3H3 antibody
    • histone 1, H3a antibody
    • Histone cluster 1, H3a antibody
    • Histone H3 3 pseudogene 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 (Human epithelial cell line from cervix adenocarcinoma) cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10 minutes. The ChIP was performed with 25 µg of chromatin, 2 µg of ab4729 (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). Primers and probes are located in the first kb of the transcribed region.

  • ab4729 staining Histone H3 (acetyl K27) in HeLa (Human epithelial cell line from cervix adenocarcinoma) cells.

    The cells were incubated with 10 mM sodium butyrate (ab120948) for 6 hours (Treated) or solvent-only for control purposes (Non-treated). Cells were fixed with 100% methanol (5 minutes) and then blocked in 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1 hour. The cells were then incubated with ab4729 at 0.5 µg/ml and ab7291 at 1 µg/ml overnight at +4°C, followed by a further incubation at room temperature for 1 hour with a anti-rabbit AlexaFluor®488 secondary antibody (ab150077) at 2 μg/ml (shown in green) and a goat anti-mouse AlexaFluor®594 (ab150120) at 2 μg/ml (shown in pseudo colour red). Nuclear DNA was labeled in blue with DAPI.

    Negative controls: 1– Rabbit primary and anti-mouse secondary antibody; 2 – Mouse primary antibody and anti-rabbit secondary antibody. Controls 1 and 2 indicate that there is no unspecific reaction between primary and secondary antibodies used.

  • IHC image of ab4729 staining Histone H3 (acetyl K27) in human colon formalin-fixed paraffin-embedded tissue sections*, performed on a Leica Bond.

    The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer pH 6 for 20 minutes. The section was then incubated with ab4729, 5 µg/ml, for 15 minutes 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.

    No primary antibody was used in the negative control (shown on the inset).

    For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

    *Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

  • HeLa (Human epithelial cell line from cervix adenocarcinoma) cells were incubated at 37°C for 6 hours with vehicle control (0 μM) and different concentrations of sodium butyrate (ab120948). Increased expression of histone H3 (acetyl K27)(ab4729) in HeLa cells correlates with an increase in sodium butyrate concentration, as described in literature.

    Whole cell lysates were prepared with RIPA buffer (containing protease inhibitors and sodium orthovanadate), 2.5 μg of each were loaded on the gel and the WB was run under reducing conditions. After transfer the membrane was blocked for an hour using 5% BSA before being incubated with ab4927 at 1 μg/ml and ab8227 at 1 μg/ml overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP (ab97051) at 1/10,000 dilution and visualised using ECL development solution.

  • All batches of ab4729 are tested in Peptide Array against peptides to different Histone H3 modifications. Six dilutions of each peptide are printed on to the Peptide Array in triplicate and results are averaged before being plotted on to a graph. Results show strong binding to Histone H3 - acetyl K27 peptide (ab24404), indicating that this antibody specifically recognises the Histone H3 - acetyl K27 modification.

    ab24404 - Histone H3 - acetyl K27

    ab15591 - Histone H3 - acetyl K14

    ab24003 - Histone H3 - acetyl K18

    ab17163 - Histone H3 unmodified

    ab48359 - Histone H3 - acetyl K23

    ab41409 - Histone H3 - acetyl K36

    ab15662 - Histone H4 - acetyl K12

    ab16635 - Histone H3 acetyl K9

  • Lanes 1 & 3 : Anti-Histone H3 (acetyl K27) antibody - ChIP Grade (ab4729) at 0.2 µg/ml
    Lanes 2 & 4 : Anti-Histone H3 (acetyl K27) antibody - ChIP Grade (ab4729) at 0.1 µg/ml

    Lanes 1-2 : Calf thymus histone lysate
    Lanes 3-4 : Calf thymus histone lysate with Human Histone H3 (acetyl K27) peptide (ab24404) at 2 µg

    Lysates/proteins at 1 µg per lane.

    Secondary
    All lanes : Goat anti-rabbit (HRP) at 1/2000 dilution

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



    ab4729 specifically recognises acetyl K27 histone H3 in catlf thymus histone lysate, which is specifically blocked using the immunizing peptide ab24404.
  • Anti-Histone H3 (acetyl K27) antibody - ChIP Grade (ab4729) at 1 µg/ml + HeLa (Human epithelial cell line from cervix adenocarcinoma) histone preparation, nuclear Lysate - Butyrate treated at 2.5 µg

    Secondary
    Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/10000 dilution

    Developed using the ECL technique.

    Performed under reducing conditions.

    Predicted band size: 15 kDa
    Additional bands at: 17 kDa. We are unsure as to the identity of these extra bands.


    Exposure time: 10 seconds


     

     

  • Primary antibody: ab4729 (H3 acetyl K27)
    Dilution: 1/100

    ab4729 strongly stained histones of mouse ES cells. However, fluroescence was greatly diminished following pre-blocking using a H3 acetyl K9 peptide. This suggests the antibody cross-reacts with the K9 and K27 residues.

     

References

This product has been referenced in:
  • Festa BP  et al. Impaired autophagy bridges lysosomal storage disease and epithelial dysfunction in the kidney. Nat Commun 9:161 (2018). Read more (PubMed: 29323117) »
  • Qin Y  et al. An obesity-associated gut microbiome reprograms the intestinal epigenome and leads to altered colonic gene expression. Genome Biol 19:7 (2018). Read more (PubMed: 29361968) »
See all 696 Publications for this product

Customer reviews and Q&As

1-10 of 66 Abreviews or Q&A

Application
Western blot
Sample
Schmidtea mediterranea Cell lysate - whole cell (Whole worm)
Gel Running Conditions
Reduced Denaturing (Invitrogen™ NuPAGE™ Novex™ 4-12% Bis-Tris Protein Gels, 1.0 mm, 10-well)
Loading amount
15 µg
Specification
Whole worm
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 Oct 22 2018

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Human Cell lysate - whole cell (liver)
Gel Running Conditions
Non-reduced Denaturing
Loading amount
10 µg
Specification
liver
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: 25°C

Abcam user community

Verified customer

Submitted Sep 21 2018

Application
ChIP
Sample
Human Cell lysate - nuclear (Kidney (Clear Cell Renal Carcinoma))
Negative control
Parental clear cell renal carcinoma cell (786-O).
Specification
Kidney (Clear Cell Renal Carcinoma)
Detection step
Other
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
Positive control
Metastatic clear cell renal carcinoma cells (M1A).

Paulo Rodrigues

Verified customer

Submitted Aug 17 2018

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
ChIP
Sample
Mouse Cell lysate - nuclear (mouse neural stem cell)
Negative control
IgG CHIP
Specification
mouse neural stem cell
Detection step
Semiquantitative PCR
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
Positive control
RELA CHIP

Abcam user community

Verified customer

Submitted Jun 15 2018

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (HEK293)
Permeabilization
Yes - 0.1% Triton X-100, 10 min RT
Specification
HEK293
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
Fixative
Paraformaldehyde

Abcam user community

Verified customer

Submitted Jun 13 2018

Application
Western blot
Sample
Mouse Cell lysate - whole cell (mouse neural stem cell)
Gel Running Conditions
Reduced Denaturing (4-12%)
Loading amount
40 µg
Specification
mouse neural stem cell
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 20°C

Abcam user community

Verified customer

Submitted Jun 12 2018

Application
Immunoprecipitation
Sample
Mouse Tissue lysate - whole (Brain)
Total protein in input
300 µg
Immuno-precipitation step
Protein A
Specification
Brain

Abcam user community

Verified customer

Submitted Jun 06 2018

Application
ChIP
Sample
Human Cell lysate - whole cell (293T)
Negative control
Control IgG
Specification
293T
Detection step
Real-time PCR
Type
Cross-linking (X-ChIP)
Duration of cross-linking step: 15 minute(s) and 0 second(s)
Specification of the cross-linking agent: PFA
Positive control
GAPDH gene

Abcam user community

Verified customer

Submitted Jun 01 2018

Application
Western blot
Sample
Mouse Tissue lysate - whole (Brain)
Gel Running Conditions
Reduced Denaturing
Loading amount
10 µg
Specification
Brain
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: 24°C

Abcam user community

Verified customer

Submitted May 15 2018

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
ChIP
Sample
Human Cell lysate - whole cell (MCF-7 cells grown in deficient DMEM for 72h)
Negative control
A nearby region of the genome that is not a known enhancer in MCF-cells
Specification
MCF-7 cells grown in deficient DMEM for 72h
Detection step
Real-time PCR
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 at room temper

Abcam user community

Verified customer

Submitted May 08 2018

1-10 of 66 Abreviews or Q&A

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