Overview

  • Product name

    Anti-Histone H3 (acetyl K9 + K14) antibody
    See all Histone H3 primary antibodies
  • Description

    Rabbit polyclonal to Histone H3 (acetyl K9 + K14)
  • Host species

    Rabbit
  • Tested applications

    Suitable for: WB, ChIP, Dot blot, ICC/IF, CHIPseqmore details
  • Species reactivity

    Reacts with: Mouse, Human
    Predicted to work with: Arabidopsis thaliana, Zebrafish, Aspergillus nidulans
  • Immunogen

    Synthetic peptide corresponding to Human Histone H3 (acetyl K9 + K14) conjugated to keyhole limpet haemocyanin.
    Database link: P68431

  • Positive control

    • ChIP: Chromatin from HeLa cells. ChIPseq: Chromatin from HeLa S3 cells. WB: HeLa histone extract. ICC/IF: NIH/3T3 cells.

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 long term. Avoid freeze / thaw cycle.
  • Storage buffer

    Preservatives: 0.05% Sodium azide, 0.05% Proclin
    Constituent: PBS
  • Concentration information loading...
  • Purity

    Affinity purified
  • Clonality

    Polyclonal
  • Isotype

    IgG
  • Research areas

Applications

Our Abpromise guarantee covers the use of ab232952 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. Predicted molecular weight: 15 kDa.
ChIP Use at an assay dependent concentration.

Use 1 - 2 µg for 1.5 x 106 cells.

Dot blot 1/20000.
ICC/IF 1/500.
CHIPseq Use 1µg for 106 cells.

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

  • ChIP assays were performed using HeLa (human epithelial cell line from cervix adenocarcinoma) cells, ab232952 against Histone H3 (acetyl K9 + K14) and optimized primer pairs for qPCR. ChIP was performed using sheared chromatin from 1.5 million cells. A titration of ab232952 consisting of 1, 2, 5 and 10 µg per ChIP experiment was analysed. IgG (5 µg/IP) was used as negative IP control. QPCR was performed using primers specific for the promoter of the ACTB gene as a positive control target and for exon 2 of the MB gene as a negative control target. The figure shows the recovery (the relative amount of immunoprecipitated DNA compared to input DNA). These results confirm the observation that acetylation of H3K9/14 is present at active promoters.

  • ChIP was performed with 1 µg ab232952 against Histone H3 (acetyl K9 + K14) on sheared chromatin from 1 million HeLa S3 cells. IgG (2 µg/IP) was used as a negative IP control. The IP’d DNA was analysed by QPCR with optimized PCR primer pairs for the promoters of the active GAPDH and c-fos genes, used as positive control targets, and the coding region of the inactive MB gene and the Sat2 satellite repeat, used as negative control targets. The IP’d DNA was subsequently analysed with an Illumina Genome Analyzer. Library preparation, cluster generation and sequencing were performed according to the manufacturer’s instructions. The 36 bp tags were aligned to the human genome using the ELAND algorithm. These results clearly show an enrichment of the H3K9/14 double acetylation at the promoters of active genes.

  • ChIP was performed with 1 µg ab232952 against Histone H3 (acetyl K9 + K14) on sheared chromatin from 1 million HeLa S3 cells. IgG (2 µg/IP) was used as a negative IP control. The IP’d DNA was analysed by QPCR with optimized PCR primer pairs for the promoters of the active GAPDH and c-fos genes, used as positive control targets, and the coding region of the inactive MB gene and the Sat2 satellite repeat, used as negative control targets. The IP’d DNA was subsequently analysed with an Illumina Genome Analyzer. Library preparation, cluster generation and sequencing were performed according to the manufacturer’s instructions. The 36 bp tags were aligned to the human genome using the ELAND algorithm. The figure shows the peak distribution along the complete sequence and a 800 kb region of the X-chromosome (A and B) and in 100 kb regions surrounding the RBM3, GAPDH and c-fos genes (C, D and E). These results clearly show an enrichment of the H3K9/14 double acetylation at the promoters of active genes.

  • Dot Blot analysis was performed to test the cross reactivity of ab232952 against Histone H3 (acetyl K9 + K14) with peptides containing other histone modifications and the unmodified H3K9/14 sequence. One hundred to 0.2 pmol of the respective peptides were spotted on a membrane. ab232952 was used at 1/20000 dilution. ab232952 shows a high specificity for the modification of interest.

  • Anti-Histone H3 (acetyl K9 + K14) antibody (ab232952) at 1/1000 dilution + HeLa (human epithelial cell line from cervix adenocarcinoma) histone extract at 15 µg

    Predicted band size: 15 kDa

  • NIH/3T3 (mouse embryo fibroblast cell line) cells stained for Histone H3 (acetyl K9 + K14) (green) using ab232931 at 1/200 dilution in ICC/IF. Cells were fixed with 4% formaldehyde for 10’ and blocked with PBS/TX-100 containing 5% normal goat serum and 1% BSA. The cells were immunofluorescently labeled with ab232952 (left) at 1/500 dilution in blocking solution followed by an anti-rabbit antibody conjugated to Alexa488. The middle panel shows staining of the nuclei with DAPI. A merge of the two stainings is shown on the right.

References

ab232952 has not yet been referenced specifically in any publications.

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