Anti-Histone H3 (acetyl K27) antibody - ChIP Grade (ab4729)

Overview

  • Product nameAnti-Histone H3 (acetyl K27) antibody - ChIP GradeSee all Histone H3 primary antibodies ...
  • Description
    Rabbit polyclonal to Histone H3 (acetyl K27) - ChIP Grade
  • Tested applicationsIHC-Fr, ICC/IF, WB, IHC-P, CHIPseq, ChIP/Chip, ChIP, PepArr more details
  • Species reactivity
    Reacts with: Mouse, Rat, Chicken, Cow, Human, Arabidopsis thaliana, Caenorhabditis elegans, Fruit fly (Drosophila melanogaster), Monkey, Plasmodium falciparum, Cyanidioschyzon merolae
    Predicted to work with: Xenopus laevis
  • Immunogen

    Synthetic peptide conjugated to KLH derived from within residues 1 - 100 of Human Histone H3, acetylated at K27.

    (Peptide available as ab24404.)

  • Positive control
    • Calf thymus histone lysate; HeLa whole cell lysate; HeLa nuclear lysate; HeLa histone prep Butyrate treated. HeLa cells - ICC/IF

Properties

  • FormLiquid
  • Storage instructionsShipped 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 bufferpH: 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...
  • PurityImmunogen affinity purified
  • Clonality Polyclonal
  • IsotypeIgG
  • 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 Notes
IHC-Fr Use at an assay dependent concentration.
ICC/IF 1/500. Can be used with paraformaldehyde- or methanol- fixed cells.
WB Use at an assay dependent concentration. Detects a band of approximately 17 kDa (predicted molecular weight: 15 kDa).Can be blocked with Human Histone H3 (acetyl K27) peptide (ab24404).
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.
IF Use a concentration of 0.5 µg/ml.
ChIP/Chip Use at an assay dependent concentration.
ChIP Use at an assay dependent concentration.
PepArr Use a concentration of 0.2 - 0.02 µg/ml.

Target

  • FunctionCore 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 similaritiesBelongs to the histone H3 family.
  • Developmental stageExpressed 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 localizationNucleus. Chromosome.
  • Target information above from: UniProt accession P68431 The UniProt Consortium
    The Universal Protein Resource (UniProt) in 2010
    Nucleic Acids Res. 38:D142-D148 (2010) .

    Information by UniProt
  • Database links
  • Alternative names
    • acetyl H3 antibody
    • H 3 antibody
    • H3 3 Like Sequence MH921 antibody
    • H3 3A antibody
    • H3 antibody
    • H3 Histone antibody
    • H3 Histone Family Member E Pseudogene antibody
    • H3.4 antibody
    • H3/A antibody
    • H3/g antibody
    • H31_HUMAN antibody
    • H3F3 antibody
    • H3FA antibody
    • H3FT antibody
    • H3t antibody
    • HIST1H3J antibody
    • HIST3H3 antibody
    • Histone cluster 1, H3a antibody
    • Histone H3 3 Pseudogene antibody
    • Histone H3.1 antibody
    • Histone H3.3 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

Anti-Histone H3 (acetyl K27) antibody - ChIP Grade images

  • ab4729 staining Histone H3 (acetyl K27) in HeLa cells. The cells were incubated with 10mM Sodium butyrate (ab120948) for 6 hours (Treated) or solvent-only for control purposes (Non-treated). Cells were fixed with 100% methanol (5min) and then blocked in 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated with ab4729 at 0.5µg/ml and ab7291 at 1µg/ml overnight at +4°C, followed by a further incubation at room temperature for 1h with an AlexaFluor®488 Goat anti-Rabbit secondary (ab150077) at 2 μg/ml (shown in green) and AlexaFluor®594 Goat anti-Mouse secondary (ab150120) at 2 μg/ml (shown in pseudo colour red). Nuclear DNA was labelled 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.

  • 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


  • 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 : 10 seconds

     HeLa cells were incubated at 37°C for 6h 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 1/10000 dilution and visualised using ECL development solution.

  • Anti-Histone H3 (acetyl K27) antibody - ChIP Grade (ab4729) at 1 µg/ml + HeLa 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
  • 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  ab4729 (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.

  • IHC image of Histone H3 (acetyl K27) 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 ab4729, 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.
  • ab4729 (1/500) staining Histone H3 (acetyl K27) in HeLa cells (green). Cells were fixed in paraformaldehyde, permeabilized with 0.5% Triton X-100/PBS and counterstained with DAPI in order to highlight the nucleus (red). For further experimental details please see abreview.

    See Abreview

  • 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.

     

  • 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

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

    Lysates/proteins at 1 µg per lane.

    Secondary
    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.
  • All lanes : Anti-Histone H3 (acetyl K27) antibody - ChIP Grade (ab4729) at 0.2 µg/ml

    Lane 1 : HeLa whole cell lysate
    Lane 2 : HeLa nuclear lysate
    Lane 3 : HeLa whole cell lysate with Human Histone H3 peptide (ab17163) at 1 µg/ml
    Lane 4 : HeLa nuclear lysate with Human Histone H3 peptide (ab17163) at 1 µg/ml
    Lane 5 : HeLa whole cell lysate with Human Histone H3 (acetyl K27) peptide (ab24404) at 1 µg/ml
    Lane 6 : HeLa nuclear lysate with Human Histone H3 (acetyl K27) peptide (ab24404) at 1 µg/ml
    Lane 7 : HeLa whole cell lysate with Human Histone H3 (acetyl K9) peptide (ab16635) at 1 µg/ml
    Lane 8 : HeLa nuclear lysate with Human Histone H3 (acetyl K9) peptide (ab16635) at 1 µg/ml
    Lane 9 : HeLa whole cell lysate with Human Histone H3 (acetyl K18) peptide (ab24003) at 1 µg/ml
    Lane 10 : HeLa nuclear lysate with Human Histone H3 (acetyl K18) peptide (ab24003) at 1 µg/ml
    Lane 11 : HeLa whole cell lysate with Human Histone H3 (asymmetric di methyl R26) peptide (ab2854) at 1 µg/ml
    Lane 12 : HeLa nuclear lysate with Human Histone H3 (asymmetric di methyl R26) peptide (ab2854) at 1 µg/ml
    Lane 13 : HeLa whole cell lysate with Histone H4 (acetyl K12) peptide (ab15662) at 1 µg/ml
    Lane 14 : HeLa nuclear lysate with Histone H4 (acetyl K12) peptide (ab15662) at 1 µg/ml

    Lysates/proteins at 20 µg per lane.

    Secondary
    Goat Anti-Rabbit IgG H&L (HRP) (ab6721) at 1/5000 dilution

    Predicted band size : 15 kDa
    Observed band size : 17 kDa (why is the actual band size different from the predicted?)
    ab4729 recognises acetyl K27 histone H3 in HeLa whole cell extracts (lane1), which is efficiently blocked using the immunizing peptide ab24404 (lane5). ab4729 is not blocked using acetyl K9 histone H3 (lane7), acetyl K18 histone H3 (lane9), dimethyl R26 histone H3 (lane 11)or acetyl K12 histone H4 (lane 13) peptides.

    However, ab4729 is partially blocked by the unmodified peptide (lane 3). This indicates that the ab4729 mainly recognises acetyl K27 histone H3 but also to a small degree recognises unmodified histone H3.

    ab4729 does not appear to detect acetyl K27 histone H3 in HeLa nuclear extracts (lanes 2,4,6,8,10,12,14).
  • ICC/IF image of ab4729 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 (ab4729, 1µg/ml) overnight at +4°C. The secondary antibody (green) was DyLight® 488 goat anti-rabbit IgG - H&L, pre-adsorbed (ab96899) used at a 1/250 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.

  • ab4729 staining Histone H3 (acetyl K27) in Cyanidioschyzon merolae cells by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with paraformaldehyde and blocked with 5% BSA for 30 minutes at 37°C. Samples were incubated with primary antibody (1/100 in PBS + 0.1% BSA) for 1 hour at 37°C. An Alexa Fluor®488-conjugated Goat anti-rabbit polyclonal (100)was used as the secondary antibody.

    See Abreview

References for Anti-Histone H3 (acetyl K27) antibody - ChIP Grade (ab4729)

This product has been referenced in:
  • Weedon MN  et al. Recessive mutations in a distal PTF1A enhancer cause isolated pancreatic agenesis. Nat Genet 46:61-4 (2014). ChIP ; Human . Read more (PubMed: 24212882) »
  • Mokry M  et al. Many inflammatory bowel disease risk Loci include regions that regulate gene expression in immune cells and the intestinal epithelium. Gastroenterology 146:1040-7 (2014). Human . Read more (PubMed: 24333384) »

See all 69 Publications for this product

Product Wall

Application Flow Cytometry
Fixation Paraformaldehyde
Permeabilization Yes - eBioscience Permeabilization Buffer
Sample Human Cell (Differentiated Haematopoietic Stem Cells)
Specification Differentiated Haematopoietic Stem Cells
Gating Strategy Isotype negative control (white)
Preparation Cell harvesting/tissue preparation method: Typsin-EDTA Cell Dissociation
Sample buffer: PBS and 10% FBS
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Submitted Feb 27 2014

Application Western blot
Loading amount 20 µg
Gel Running Conditions Reduced Denaturing
Sample Human Tissue lysate - whole (Patient Dermal Fibroblasts)
Specification Patient Dermal Fibroblasts
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 22°C
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Submitted Feb 01 2014

Application Immunocytochemistry/ Immunofluorescence
Blocking step BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: 25°C
Sample Mouse Cell (primary hepatoctyes)
Specification primary hepatoctyes
Permeabilization No
Fixative Paraformaldehyde
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Submitted Jan 13 2014

Application ChIP
Detection step Real-time PCR
Sample Mouse Cell lysate - whole cell (Mouse ES cells)
Specification Mouse ES cells
Negative control Isotype IgG
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 Known H3K27Ac-enriched region was assessed
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Submitted Dec 13 2013

Application Western blot
Loading amount 20 µg
Gel Running Conditions Non-reduced Denaturing (15)
Sample Mouse Cell lysate - whole cell (pmHep)
Specification pmHep
Blocking step I-Block buffer as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 2µg/mL · Temperature: 25°C
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Submitted Dec 13 2013

Application ChIP
Detection step Real-time PCR
Sample Mouse Cell lysate - whole cell (pmHep)
Specification pmHep
Negative control IgG
Type Cross-linking (X-ChIP)
Duration of cross-linking step: 10 minute(s) and 0 second(s)
Specification of the cross-linking agent: Formaldehyde
Positive control no
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Submitted Dec 11 2013

Application Western blot
Loading amount 20 µg
Gel Running Conditions Reduced Denaturing (12)
Sample Human Cell lysate - whole cell (Hela cells)
Specification Hela cells
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 22°C
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Submitted Oct 22 2013

Application Western blot
Loading amount 50 µg
Gel Running Conditions Reduced Denaturing (12)
Sample Caenorhabditis elegans Tissue lysate - nuclear (C. elegan nuclear lysate)
Specification C. elegan nuclear lysate
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 22°C
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Submitted Oct 14 2013

Application Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Blocking step Serum as blocking agent for 45 minute(s) · Concentration: 1.5% · Temperature: 25°C
Antigen retrieval step Enzymatic
Sample Rat Tissue sections (GONAD)
Specification GONAD
Permeabilization No
Fixative Formaldehyde
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Submitted Sep 25 2013

Application ChIP
Detection step Real-time PCR
Sample Human Cell lysate - other (Epithelial cells)
Specification Epithelial 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
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Submitted Sep 12 2013

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