Anti-Histone H3 (acetyl K56) antibody [EPR996Y] (ab76307)

Overview

  • Product nameAnti-Histone H3 (acetyl K56) antibody [EPR996Y]
    See all Histone H3 primary antibodies
  • Description
    Rabbit monoclonal [EPR996Y] to Histone H3 (acetyl K56)
  • Tested applicationsWB, IHC-P, ICC/IFmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Human, Saccharomyces cerevisiae, African Green Monkey
  • Immunogen

    Synthetic peptide (the amino acid sequence is considered to be commercially sensitive) within Human Histone H3 aa 50 to the C-terminus (acetyl K56). The exact sequence is proprietary.

  • Positive control
    • WB: C6, HeLa and NIH/3T3 cell lysates, untreated or treated with Trichostatin A. ICC/IF: HeLa cells. IHC-P: Human breast cancer, cervical carcinoma, gastric adenocarcinoma, lung adenocarcinoma and lymphoma tissues. Mouse and rat liver tissue.
  • General notes

    This product is a recombinant rabbit monoclonal antibody.

    We are constantly working hard to ensure we provide our customers with best in class antibodies. As a result of this work we are pleased to now offer this antibody in purified format. We are in the process of updating our datasheets. The purified format is designated ‘PUR’ on our product labels. If you have any questions regarding this update, please contact our Scientific Support team.

    Produced using Abcam’s RabMAb® technology. RabMAb® technology is covered by the following U.S. Patents, No. 5,675,063 and/or 7,429,487.

     

    Alternative versions available:

    Anti-Histone H3 (acetyl K56) antibody (Alexa Fluor® 488) [EPR996Y] (ab200335)

    Anti-Histone H3 (acetyl K56) antibody (Alexa Fluor® 647) [EPR996Y] (ab200337)

Properties

  • FormLiquid
  • Storage instructionsShipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C. Avoid freeze / thaw cycle.
  • Storage bufferpH: 7.20
    Preservative: 0.01% Sodium azide
    Constituents: 59% PBS, 40% Glycerol, 0.05% BSA
  • Concentration information loading...
  • PurityProtein A purified
  • ClonalityMonoclonal
  • Clone numberEPR996Y
  • IsotypeIgG
  • Research areas

Applications

Our Abpromise guarantee covers the use of ab76307 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
WB 1/1000 - 1/2000. Detects a band of approximately 15 kDa (predicted molecular weight: 15 kDa).
IHC-P 1/100 - 1/400. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

See protocols (link: http://www.abcam.com/protocols/ihc-antigen-retrieval-protocol).

Use of HRP-conjugated or polymerized HRP secondary antibodies, stronger signals have been found using the polymerized HRP secondary.

ICC/IF 1/100 - 1/250.

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.
  • Information by UniProt
  • Database links
  • Alternative names
    • H 3 antibody
    • H3 antibody
    • H3/A antibody
    • H31_HUMAN antibody
    • H3FA antibody
    • HIST1H3J antibody
    • Histone cluster 1, H3a 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 K56) antibody [EPR996Y] images

  • All lanes : Anti-Histone H3 (acetyl K56) antibody [EPR996Y] (ab76307) at 1/2000 dilution (purified)

    Lane 1 : NIH/3T3 whole cell lysate - untreated
    Lane 2 : NIH/3T3 whole cell lysate - treated with Trichostatin A (500/ng/ml, 4hr)

    Lysates/proteins at 10 µg per lane.

    Secondary
    Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

    Predicted band size : 15 kDa
    Observed band size : 15 kDa

    Blocking and dilution buffer: 5% NFDM /TBST.

  • All lanes : Anti-Histone H3 (acetyl K56) antibody [EPR996Y] (ab76307) at 1/2000 dilution (purified)

    Lane 1 : C6 whole cell lysate - untreated
    Lane 2 : C6 whole cell lysate - treated with Trichostatin A (500ng/ml, 4hr)

    Lysates/proteins at 10 µg per lane.

    Secondary
    Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

    Predicted band size : 15 kDa
    Observed band size : 15 kDa

    Blocking and dilution buffer: 5% NFDM /TBST.

  • All lanes : Anti-Histone H3 (acetyl K56) antibody [EPR996Y] (ab76307) at 1/2000 dilution (purified)

    Lane 1 : HeLa whole cell lysate - untreated
    Lane 2 : HeLa whole cell lysate - treated with Trichostatin A (500ng/ml, 4hr)

    Lysates/proteins at 10 µg per lane.

    Secondary
    Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

    Predicted band size : 15 kDa
    Observed band size : 15 kDa

    Blocking and dilution buffer: 5% NFDM /TBST.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of rat liver tissue labelling Histone H3 (acetyl K56) with purified ab76307 at 1/400. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of mouse liver tissue labelling Histone H3 (acetyl K56) with purified ab76307 at 1/400. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human cervical carcinoma tissue labelling Histone H3 (acetyl K56) with purified ab76307 at 1/400. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

  • Immunocytochemistry/Immunofluorescence analysis of HeLa cells (+/- TSA, 500 ng/ml, 4hr) labelling Histone H3 (acetyl K56) with purified ab76307 at 1/100. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. ab150077, an Alexa Fluor® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody. DAPI (blue) was used as the nuclear counterstain. ab7291, a mouse anti-tubulin (1/1000) and ab150120, an Alexa Fluor® 594-conjugated goat anti-mouse IgG (1/1000) were also used.

    Control 1: primary antibody (1/100) and secondary antibody, ab150120, an Alexa Fluor® 594-conjugated goat anti-mouse IgG (1/1000).

    Control 2: ab7291 (1/1000) and secondary antibody, ab150077, an Alexa Fluor® 488-conjugated goat anti-rabbit IgG (1/1000).

  • Unpurified ab76307 staining Histone H3 (HEK56 Ac) in African Green Monkey kidney epithelial (CV-1 line) cell by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with paraformaldehyde, permeabilized with Triton and blocked with 5% BSA for 1 hour at 21°C. Samples were incubated with primary antibody (1/500) for 16 hour at 4°C.

    CV-1 cells were infected with Simian virus 40 (SV40). 32 hours later the cells were incubated with EdU for 30 minutes before pre-extraction to detect active DNA replication foci and fixed, stained, and visualized by confocal microscopy.
    Blue - Hoechst stain, red - anti-T-Ag, magenta - EdU, green - anti-H3K56 Ac. Bar, 5 µm.

    See Abreview

  • Immunocytochemistry/Immunofluorescence analysis of HeLa cells labelling Histone H3 (acetyl K56) with unpurified ab76307 at a dilution of 1/100.

  • All lanes : Anti-Histone H3 (acetyl K56) antibody [EPR996Y] (ab76307) at 1/2000 dilution (unpurified)

    Lane 1 : C6 cell lysate, untreated
    Lane 2 : C6 cell lysate, treated with TSA

    Lysates/proteins at 10 µg per lane.

    Secondary
    HRP-conjugated goat anti-rabbit IgG at 1/1000 dilution

    Predicted band size : 15 kDa
    Observed band size : 15 kDa
  • IHC image of unpurified ab76307 staining in human breast cancer formalin fixed paraffin embedded tissue section, performed on a Leica Bond™ 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 ab76307, undiluted, for 15 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.
    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.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human cervical carcinoma tissue labelling Histone H3 (acetyl K56) with unpurified ab76307.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human gastric adenocarcinoma tissue labelling Histone H3 (acetyl K56) with unpurified ab76307.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human lung adenocarcinoma tissue labelling Histone H3 (acetyl K56) with unpurified ab76307.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human lymphoma tissue labelling Histone H3 (acetyl K56) with unpurified ab76307.

References for Anti-Histone H3 (acetyl K56) antibody [EPR996Y] (ab76307)

This product has been referenced in:
  • Benard A  et al. Nuclear expression of histone deacetylases and their histone modifications predicts clinical outcome in colorectal cancer. Histopathology 66:270-82 (2015). Read more (PubMed: 25307864) »
  • Zhang J  et al. SOX4 inhibits GBM cell growth and induces G0/G1 cell cycle arrest through Akt-p53 axis. BMC Neurol 14:207 (2014). WB ; Human . Read more (PubMed: 25366337) »

See all 18 Publications for this product

Product Wall

Application Western blot
Loading amount 20 µg
Gel Running Conditions Reduced Denaturing
Sample Xenopus laevis Cell lysate - other (Egg extracts and chromatin)
Specification Egg extracts and chromatin
Blocking step Milk as blocking agent for 30 minute(s) · Concentration: 5% · Temperature: 22°C
Username

Abcam user community

Verified customer

Submitted May 01 2014

Abcam guarantees this product to work in the species/application used in this Abreview.
Application Immunocytochemistry/ Immunofluorescence
Blocking step BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 21°C
Sample African Green Monkey Cell (epithel kidney CV-1)
Specification epithel kidney CV-1
Permeabilization Yes - triton
Fixative Paraformaldehyde
Username

Herr Dr. Gabor Rohaly

Verified customer

Submitted Jul 01 2013

>Below please find the IF protocol we suggest:


Solutions and reagents

1.1. Washing buffer:
PBST washing buffer: 1X PBS / 0.1% Tween-20


1.2. 2% Paraformaldehyde (prepare fresh by dissolving paraformaldehyde in 1...

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