Recombinant
RabMAb

Anti-Histone H3 (acetyl K9) antibody [EPR16988] - ChIP Grade (ab177177)

Overview

  • Product name
    Anti-Histone H3 (acetyl K9) antibody [EPR16988] - ChIP Grade
    See all Histone H3 primary antibodies
  • Description
    Rabbit monoclonal [EPR16988] to Histone H3 (acetyl K9) - ChIP Grade
  • Host species
    Rabbit
  • Tested applications
    Suitable for: ICC/IF, IHC-P, WB, PepArr, ChIPmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Human
  • Immunogen

    Synthetic peptide within Human Histone H3 aa 1-100 (acetyl K9). The exact sequence is proprietary.
    Database link: P68431

  • Positive control
    • WB: Whole cell lysates from HeLa and NIH/3T3 cells treated with Trichostatin A. IHC-P: Human colon, Mouse colon and Rat spleen tissues. ICC/IF: HeLa cells.
  • General notes

     

     

    Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMab® patents.

    This product is a recombinant rabbit monoclonal antibody.

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
    Preservative: 0.01% Sodium azide
    Constituents: 59% PBS, 40% Glycerol, 0.05% BSA
  • Concentration information loading...
  • Purity
    Protein A purified
  • Clonality
    Monoclonal
  • Clone number
    EPR16988
  • Isotype
    IgG
  • Research areas

Applications

Our Abpromise guarantee covers the use of ab177177 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
ICC/IF 1/3000.
IHC-P 1/200. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
WB 1/5000. Detects a band of approximately 15 kDa (predicted molecular weight: 15 kDa).
PepArr Use at an assay dependent concentration.
ChIP Use 2 µg for 25 µg of chromatin.

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

  • All lanes : Anti-Histone H3 (acetyl K9) antibody [EPR16988] - ChIP Grade (ab177177) at 1/1000 dilution

    Lane 1 : HeLa treated with Trichostatin A 500 ng/ml for 4hr whole cell lysates
    Lane 2 : Untreated HeLa whole cell lysates
    Lane 3 : NIH/3T3 treated with Trichostatin A 500 ng/ml for 4hr whole cell lysates
    Lane 4 : Untreated NIH/3T3 whole cell lysates

    Lysates/proteins at 10 µg per lane.

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

  • Chromatin was prepared from HeLa cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10 minutes. The ChIP was performed with 25µg of chromatin, 2µg of ab177177 (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 for active and inactive loci, Sybr green approach for heterochromatic loci). Primers and probes are located in the first kb of the transcribed region.

  • Immunohistochemical analysis of paraffin-embedded Human colon tissue labeling Histone H3 (acetyl K9) with ab177177 at 1/500 dilution, followed by Goat Anti-Rabbit HRP (IgG H&L) (ab97051) at 1/500 dilution. Nuclear staining on glandular epithelium of Human colon tissue is observed. Counter stained with Hematoxylin.

    Negative control: PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

  • ab177177 was tested in Peptide Array against 501 different modified and unmodified histone peptides; each peptide is printed on the array at six concentrations (each in triplicate).
    Circle area represents affinity between the antibody and a peptide: all antigen-containing peptides are displayed as red circles, all other peptides as blue circles. The affinity is calculated as area under curve when antibody binding values are plotted against the corresponding peptide concentration. Each circle area is normalized to the peptide with the strongest affinity.
    The complete dataset, including full list of all peptides and information on the position of each peptide in the diagram, can be downloaded here.

  • Immunohistochemical analysis of paraffin-embedded mouse colon tissue labeling Histone H3 (acetyl K9) with ab177177 at 1/500 dilution, followed by Goat Anti-Rabbit HRP (IgG H&L) (ab97051) at 1/500 dilution. Nuclear staining on glandular epithelium of mouse colon tissue is observed. Counter stained with Hematoxylin.

    Negative control: PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

  • Immunohistochemical analysis of paraffin-embedded rat spleen tissue labeling Histone H3 (acetyl K9) with ab177177 at 1/500 dilution, followed by Goat Anti-Rabbit HRP (IgG H&L) (ab97051) at 1/500 dilution. Nuclear staining on rat spleen tissue is observed. Counter stained with Hematoxylin.

    Negative control: PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

  • Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa cells labeling Histone H3 (acetyl K9) with ab177177 at 1/3000 dilution, followed by Goat anti-rabbit Alexa Fluor® 488 (IgG) (ab150077) secondary antibody at 1/400 dilution (green). Confocal image shows nuclear staining on HeLa cell line. The expression increased after treatment with Trichostatin A (500 ng/ml) for 4 hours. The nuclear counter stain is DAPI (blue). Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/500 dilution and ab150120 (goat anti-mouse AlexaFluor®594 secondary antibody) at 1/500 dilution (red).
    The negative controls are as follows:
    -ve control 1: ab177177 at 1/3000 dilution followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution.
    -ve control 2: ab7291 (anti-Tubulin mouse mAb) at 1/500 dilution followed by ab150077 (Alexa Fluor®488 Goat Anti-Rabbit IgG H&L) at 1/400 dilution.

References

This product has been referenced in:
  • Kulej K  et al. Time-resolved Global and Chromatin Proteomics during Herpes Simplex Virus Type 1 (HSV-1) Infection. Mol Cell Proteomics 16:S92-S107 (2017). Read more (PubMed: 28179408) »
See 1 Publication for this product

Customer reviews and Q&As

Application
Western blot
Sample
Human Cell lysate - whole cell (MCF7)
Gel Running Conditions
Reduced Denaturing (15%)
Loading amount
10 µg
Treatment
TNF alpha 20ng/ml for 1 hr
Specification
MCF7
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 22°C

Ifeoluwa Adewumi

Verified customer

Submitted Feb 28 2017

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

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