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Products:Epigenetics and Nuclear Signaling >> Histones >> H3 >> Acetylated
Overview
Product name
Anti-Histone H3 (acetyl K27) antibody - ChIP Grade
See all Histone H3 products (24) ...
Description
Rabbit polyclonal to Histone H3 (acetyl K27) - ChIP Grade
Specificity
ab4729 recognises acetyl K27 histone H3 in western blot and immunofluorescence. However, it may also recognise to a small degree acetyl K9 histone H3 (immunofluorescence) and unmodified histone H3 (western blot). See figures below.
Tested applications
ICC/IF, WB, IHC-P, CHIPseq, ChIP/Chip, ChIP, IFmore details
Cross reactivity
Reacts with
Mouse, Cow, Human, Arabidopsis thaliana, Fruit fly (Drosophila melanogaster)
Predicted to work with
Xenopus laevis, Caenorhabditis elegans
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.
Properties
Form
Liquid
Storage instructions
Store at +4°C short term (1-2 weeks). Aliquot and store at -20°C or -80°C. Avoid repeated freeze / thaw cycles.
Storage buffer
Preservative: 0.02% Sodium Azide
Constituents: 1% BSA, PBS. pH 7.4
Concentration
Concentration information loading...
Purity
Immunogen affinity purified
Clonality
Polyclonal
Isotype
IgG
Research areas
Epigenetics and Nuclear Signaling >> ChIP'ing antibodies >> ChIP'ing antibodies
Epigenetics and Nuclear Signaling >> Histones >> H3 >> Acetylated
Applications
Show applications key
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.
ICC/IF: 1/500(Can be used with paraformaldehyde- or methanol- fixed cells.)
WB: 1/1000Detects a band of approximately 17 kDa.
IHC-P: Use a concentration of 1 µg/mlPerform heat mediated antigen retrieval before commencing with IHC staining protocol.
CHIPseq: Use at an assay dependent dilution.
ChIP/Chip: Use at an assay dependent dilution.
ChIP: Use at an assay dependent concentration.
IF: 1/300
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.
Target information above from: UniProt accessionP68431
The UniProt Consortium
The Universal Protein Resource (UniProt) in 2010
Nucleic Acids Res. 38:D142-D148 (2010).
Alternative names
- H3FA antibody
- acetyl H3 antibody
- H 3 antibody
see all
Database links
- Entrez Gene: 8350 Human
- Entrez Gene: 8351 Human
- Entrez Gene: 8352 Human
- Entrez Gene: 8353 Human
- Entrez Gene: 8354 Human
- Entrez Gene: 8355 Human
- Entrez Gene: 8356 Human
- Entrez Gene: 8357 Human
see all
Anti-Histone H3 (acetyl K27) antibody - ChIP Grade images:
ChIP - Histone H3 (acetyl K27) antibody - ChIP Grade (ab4729)
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.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Histone H3 (acetyl K27) antibody - ChIP Grade (ab4729)
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.
Western blot - Histone H3 (acetyl K27) antibody - ChIP Grade (ab4729)
All lanes : Anti-Histone H3 (acetyl K27) antibody - ChIP Grade (ab4729) at 1 µg/ml
Lane 1 : HeLa Histone Preparation Nuclear Lysate - Butyrated
Lane 2 : HeLa Histone Preparation Nuclear Lysate - Butyrated with Histone H3 peptide - unmodified R17 at 0.5 µg/ml
Lane 3 : HeLa Histone Preparation Nuclear Lysate - Butyrated with Histone H3 peptide - acetyl K9 (ab16635) at 0.5 µg/ml
Lane 4 : HeLa Histone Preparation Nuclear Lysate - Butyrated with Histone H3 peptide - acetyl K14 at 0.5 µg/ml
Lane 5 : HeLa Histone Preparation Nuclear Lysate - Butyrated with Histone H3 peptide - acetyl K18 (ab24003) at 0.5 µg/ml
Lane 6 : HeLa Histone Preparation Nuclear Lysate - Butyrated with Histone H3 peptide - acetyl K23 (ab48359) at 0.5 µg/ml
Lane 7 : HeLa Histone Preparation Nuclear Lysate - Butyrated with Histone H3 peptide - acetyl K27 (ab24404) at 0.5 µg/ml
Lane 8 : HeLa Histone Preparation Nuclear Lysate - Butyrated with Histone H3 peptide - acetyl K36 at 0.5 µg/ml
Lane 9 : HeLa Histone Preparation Nuclear Lysate - Butyrated with Histone H4 peptide - acetyl K12 (ab15662) at 0.5 µg/ml
Lysates/proteins at 2.5 µg per lane.
Secondary
Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution
Performed under reducing conditions.
Predicted band size : 15.4 kDa
Observed band size : 17 kDa (why is the actual band size different from the predicted?)
Additional bands at : 12 kDa. We are unsure as to the identity of these extra bands.
Exposure time : 150 seconds
Immunocytochemistry/ Immunofluorescence - Anti-Histone H3 (acetyl K27) antibody - ChIP Grade (ab4729)
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.
Image courtesy of an Abreview submitted by Dr. Kirk McManus, Univ. of Manitoba/Cancer Care MICB, Canada
Immunofluorescence - Histone H3 (acetyl K27) antibody (ab4729)
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.
Petra Hajkova - Gurdon Institute, Cambridge University
Western blot - Histone H3 (acetyl K27) antibody (ab4729)
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 Histone H3 peptide - acetyl K27 (ab24404) at 2 µg
Lane 4 : Calf thymus histone lysate with Histone H3 peptide - acetyl K27 (ab24404) at 2 µg
Lysates/proteins at 1 µg per lane.
Secondary
Goat anti-rabbit (HRP) at 1/2000 dilution
Predicted band size : 15.4 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.
Western blot - Histone H3 (acetyl K27) antibody (ab4729)
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 Histone H3 peptide (ab17163) at 1 µg/ml
Lane 4 : HeLa nuclear lysate with Histone H3 peptide (ab17163) at 1 µg/ml
Lane 5 : HeLa whole cell lysate with Histone H3 peptide - acetyl K27 (ab24404) at 1 µg/ml
Lane 6 : HeLa nuclear lysate with Histone H3 peptide - acetyl K27 (ab24404) at 1 µg/ml
Lane 7 : HeLa whole cell lysate with Histone H3 peptide - acetyl K9 (ab16635) at 1 µg/ml
Lane 8 : HeLa nuclear lysate with Histone H3 peptide - acetyl K9 (ab16635) at 1 µg/ml
Lane 9 : HeLa whole cell lysate with Histone H3 peptide - acetyl K18 (ab24003) at 1 µg/ml
Lane 10 : HeLa nuclear lysate with Histone H3 peptide - acetyl K18 (ab24003) at 1 µg/ml
Lane 11 : HeLa whole cell lysate with Histone H3 peptide - asymmetric di methyl R26 (ab2854) at 1 µg/ml
Lane 12 : HeLa nuclear lysate with Histone H3 peptide - asymmetric di methyl R26 (ab2854) at 1 µg/ml
Lane 13 : HeLa whole cell lysate with Histone H4 peptide - acetyl K12 (ab15662) at 1 µg/ml
Lane 14 : HeLa nuclear lysate with Histone H4 peptide - acetyl K12 (ab15662) at 1 µg/ml
Lysates/proteins at 20 µg per lane.
Secondary
Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (HRP) (ab6721) at 1/5000 dilution
Predicted band size : 15.4 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).
Immunocytochemistry/ Immunofluorescence - Histone H3 (acetyl K27) antibody - ChIP Grade (ab4729)
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.
References for Anti-Histone H3 (acetyl K27) antibody - ChIP Grade (ab4729)
This product has been referenced in:
- Harismendy Oet al. 9p21 DNA variants associated with coronary artery disease impair interferon-? signalling response. Nature 470:264-8 (2011). ChIP; Human.Read more (PubMed: 21307941) »
- Kim YW & Kim A Characterization of histone H3K27 modifications in the ß-globin locus. Biochem Biophys Res Commun 405:210-5 (2011). ChIP; Human.Read more (PubMed: 21219849) »
See all 11 publications for this product
Publishing research using ab4729? Please let us know so that we can cite the reference in this datasheet
Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"