Anti-Histone H3 antibody - ChIP Grade (ab12079)

Overview

  • Product name
    Anti-Histone H3 antibody - ChIP Grade
    See all Histone H3 primary antibodies
  • Description
    Goat polyclonal to Histone H3 - ChIP Grade
  • Tested applications
    Suitable for: ChIP, IP, ChIP/Chip, WB, IHC-Pmore details
  • Species reactivity
    Reacts with: Mouse, Chicken, Dog, Human, Saccharomyces cerevisiae, Xenopus laevis, Drosophila melanogaster, Chinese hamster
    Predicted to work with: Arabidopsis thaliana, Caenorhabditis elegans, Schizosaccharomyces pombe
  • Immunogen

    Synthetic peptide corresponding to Human Histone H3 aa 100 to the C-terminus.
    Database link: P68431
    (Peptide available as ab12149)

  • Positive control
    • HeLa Cell Histone preparation IHC: FFPE human testis tissue sections.
  • General notes

     This antibody is raised using the same immunogen as our popular anti-Histone H3 rabbit polyclonal - ab1791

Properties

Applications

Our Abpromise guarantee covers the use of ab12079 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
ChIP Use 2-4 µg for 25 µg of chromatin. Every new batch of this antibody is tested at Abcam in ChIP.
IP Use at an assay dependent concentration.
ChIP/Chip Use at an assay dependent concentration.
WB Use a concentration of 1 µg/ml. Detects a band of approximately 17 kDa (predicted molecular weight: 15 kDa).Can be blocked with Human Histone H3 peptide (ab12149).
IHC-P Use a concentration of 5 µg/ml. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

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

Anti-Histone H3 antibody - ChIP Grade images

  • Chromatin was prepared from U2OS cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10 min. The  ChIP was performed with 25 µg of chromatin, 2 µg of  ab12079 (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 for active and inactive loci, Sybr green approach for heterochromatic loci). Primers and probes are located in the first kb of the transcribed region.

  • IHC image of Histone H3 staining in human testis formalin fixed paraffin embedded tissue section, performed on a Leica Bond system using the standard protocol B. 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 ab12079, 5µg/ml, for 15 mins at room temperature. A donkey anti-goat biotinylated secondary antibody was used to detect the primary, and visualized using an HRP conjugated ABC 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.

  • Anti-Histone H3 antibody - ChIP Grade (ab12079) at 0.5 µg/ml + HeLa Cell Histone prep at 5 µg

    Secondary
    Rabbit Anti-Goat IgG H&L (HRP) (ab6741) at 1/5000 dilution

    Performed under reducing conditions.

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

    Histone H3 antibody (ab12079) at 0.5 ug/ml, HeLa Cell Histone prep at 5 ug

    Secondary
    Rabbit polyclonal to Goat IgG H&L (HRP) (ab6741) at 1/5000 dilution

    Performed under reducing conditions.

    Predicted band size : 15kD

  • All lanes : Anti-Histone H3 antibody - ChIP Grade (ab12079) at 1 µg/ml

    Lane 1 : Chicken S1:29 Cell lysate at 10 µg
    Lane 2 : CHO Cell lysate at 10 µg
    Lane 3 : Dog MDCK Cell lysate at 10 µg
    Lane 4 : Mouse 3T3 Whole Cell Lysate
    Lane 5 : Xenopus embryo Cell lysate at 10 µg

    Secondary
    Alexa Fluor 680 Rabbit anti-Goat IgG at 1/10000 dilution

    Performed under reducing conditions.

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

References for Anti-Histone H3 antibody - ChIP Grade (ab12079)

This product has been referenced in:

See all 11 Publications for this product

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Read More
Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Saccharomyces cerevisiae Cell lysate - whole cell (S288c)
Loading amount
25 µg
Specification
S288c
Gel Running Conditions
Reduced Denaturing (15% gel)
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: RT°C
Username

Mr. Harsh Ja

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 (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
Username

Mr. Harsh Ja

Verified customer

Submitted Aug 02 2011

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
ChIP on 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
Microarray
Negative control
No antibody control
Username

Abcam user community

Verified customer

Submitted May 31 2011

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunoprecipitation
Sample
Saccharomyces cerevisiae Cell lysate - nuclear (W303 cells)
Total protein in input
10 µg
Specification
W303 cells
Treatment
1% MMS Methyl methanesulfonate (DNA damaging agent)
Immuno-precipitation step
Protein A/G
Username

Abcam user community

Verified customer

Submitted May 27 2011

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
ChIP
Sample
Saccharomyces cerevisiae Cell lysate - nuclear (BY4741 cells)
Specification
BY4741 cells
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
Username

Abcam user community

Verified customer

Submitted May 26 2011

Abcam has not validated the combination of species/application used in this Abreview.
Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (Human Fetal Lung Fibroblasts)
Specification
Human Fetal Lung Fibroblasts
Fixative
Formaldehyde
Permeabilization
Yes - Triton-X 0.3%
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
Username

Abcam user community

Verified customer

Submitted May 19 2011

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Saccharomyces cerevisiae Cell lysate - whole cell (Yeast whole cell extract)
Loading amount
10 µg
Specification
Yeast whole cell extract
Gel Running Conditions
Reduced Denaturing (15% gel)
Blocking step
Milk as blocking agent for 18 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 4°C
Username

Abcam user community

Verified customer

Submitted May 19 2011

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

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