Anti-Histone H3 (acetyl K9 + K14 + K18 + K23 + K27) antibody - ChIP Grade (ab47915)

Overview

  • Product name
    Anti-Histone H3 (acetyl K9 + K14 + K18 + K23 + K27) antibody - ChIP Grade
    See all Histone H3 primary antibodies
  • Description
    Rabbit polyclonal to Histone H3 (acetyl K9 + K14 + K18 + K23 + K27) - ChIP Grade
  • Host species
    Rabbit
  • Specificity

    This antibody recognizes histone H3 acetylated at lysines 9, 14, 18, 23 or 27 as confirmed by dot blot with non-modified histone H3 peptide or peptides No reaction with non-modified histone H3 peptide as tested by dot blot.

  • Tested applications
    Suitable for: ChIP, ICC/IF, WBmore details
  • Species reactivity
    Reacts with: Human
    Predicted to work with: Mouse
  • Immunogen

    Synthetic peptide corresponding to Human Histone H3 (N terminal) (acetyl K9 + K14 + K18 + K23 + K27).

  • Positive control
    • WB: HeLa cell lysate. ICC/IF: HeLa cells. ChIP: Chromatin prepared from HeLa cells.
  • General notes

    Learn about ChIP assay kits, other ChIP antibodies, protocols and more in the ChIP assay guide.

Properties

Applications

Our Abpromise guarantee covers the use of ab47915 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 at an assay dependent concentration. PubMed: 20080953

Use 10 µL per ChIP experiment

ICC/IF 1/1000.
WB 1/500 - 1/5000. Detects a band of approximately 17 kDa (predicted molecular weight: 15 kDa).

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

  • HP1α delocalises from chromocentres upon proteasome inhibition whereas the histone modifications remain unaffected.

    (A) Proteasome inhibition does not affect canonical histone modifications on major and minor satellite repeats. NIH/3T3 (Mouse embryo fibroblast cell line) cells were treated with 20μM MG132 for 4h followed by ChIP–qPCR analysis using antibodies against repressive mark H3K9me3 and activating marks H3K4me3, H3K36me3, and H3ac (ab47915). Data is shown as relative enrichment to H3 with background subtraction. The y-axis scale was adjusted depending on the signal obtained with different antibodies. Error bars = SEM of 3 biological replicates.

  • ChIP was performed using a high sensitivity kit with 5 µg of chromatin from HeLa (Human epithelial cell line from cervix adenocarcinoma) cells and 10 µL of Anti-Histone H3 (acetyl K9 + K14 + K18 + K23 + K27) antibody ab47915.

    ChIP DNA was used in qPCR with the negative control primer pairs or gene-specific primer pairs as indicated. Data are presented as binding events detected per 1000 cells using a normalization scheme which accounts for primer efficiency and the amount of chromatin used in the ChIP reaction.

  • All lanes : Anti-Histone H3 (acetyl K9 + K14 + K18 + K23 + K27) antibody - ChIP Grade (ab47915) at 1/2000 dilution

    Lane 1 : HeLa (Human epithelial cell line from cervix adenocarcinoma) cells, no treatment
    Lane 2 : HeLa cells, treatment with sodium butyrate

    Lysates/proteins at 10 µg per lane.

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

  • ICC/IF image of ab47915 stained HeLa (Human epithelial cell line from cervix adenocarcinoma) cells.

    The cells were fixed in 100% methanol (5 min) and then incubated in 1% BSA / 10% normal goat serum ab7481 / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilize the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab47915, 1/1000 dilution) overnight at +4°C. The secondary antibody (green) was ab96899, a goat anti-rabbit DyLight® 488 (IgG; H+L) 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 1.43 µM.

  • ab47915 tested by dot blot analysis.
    Dot blot analysis was used to confirm the specificity of ab47915. Acetylated peptides corresponding to the immunogen and related peptides were spotted onto PVDF and probed with the antibody at a dilution of 1:1,000. The amount of peptide (picomoles) spotted is indicated next to the row.
    Lane 1: acetyl-Lys9 peptide. Lane 2: unmodified Lys9 peptide. Lane 3: acetyl-Lys14 peptide. Lane 4: unmodified Lys14 peptide. Lane 5: acetyl-Lys18 peptide. Lane 6: unmodified Lys18 peptide. Lane 7: acetyl-Lys23 peptide. Lane 8: unmodified Lys23 peptide. Lane 9: acetyl-Lys27 peptide. Lane 10: unmodified Lys27 peptide.

References

This product has been referenced in:
  • Smith ER  et al. TGF-ß1 modifies histone acetylation and acetyl-coenzyme A metabolism in renal myofibroblasts. Am J Physiol Renal Physiol N/A:N/A (2019). Read more (PubMed: 30623724) »
  • Park HJ  et al. HOS15 Interacts with the Histone Deacetylase HDA9 and the Evening Complex to Epigenetically Regulate the Floral Activator GIGANTEA. Plant Cell 31:37-51 (2019). Read more (PubMed: 30606777) »
See all 38 Publications for this product

Customer reviews and Q&As

1-6 of 6 Abreviews or Q&A

Application
Western blot
Sample
Mouse Cell lysate - whole cell (C2C12 myotubes)
Gel Running Conditions
Reduced Denaturing (15%)
Loading amount
35 µg
Treatment
TSA 0.5 uM for 24h
Specification
C2C12 myotubes
Blocking step
Rockland blocking buffer cat #M13-070 as blocking agent for 30 minute(s) · Concentration: 100% · Temperature: 24°C

Liping Zhang

Verified customer

Submitted Feb 26 2019

Application
ChIP
Sample
Sheep Cell lysate - whole cell (Ovine B cell line)
Negative control
Ip IgG
Specification
Ovine B cell line
Detection step
Real-time PCR
Type
Cross-linking (X-ChIP)
Duration of cross-linking step: 10 minute(s) and 0 second(s)
Specification of the cross-linking agent: Formaldéhyde 1%
Positive control
Regions 1 to 3 that are known to be epigenetically positive

Mrs. Caroline Vanhulle

Verified customer

Submitted Dec 02 2016

Application
Immunohistochemistry (Resin sections)
Sample
Astatotilapia burtoni Tissue sections (Brain Preoptica area LRWhite embedded)
Specification
Brain Preoptica area LRWhite embedded

Sebastian Alvarado

Verified customer

Submitted May 04 2016

Application
Western blot
Sample
Astatotilapia burtoni Cell lysate - nuclear (Brain)
Gel Running Conditions
Reduced Denaturing (4-20%)
Loading amount
25 µg
Specification
Brain
Blocking step
Fluorescent blocking buffer (MB-070 Rockland) as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 100% · Temperature: 25°C

Sebastian Alvarado

Verified customer

Submitted Apr 27 2016

Answer



I asked the lab what they used for the testing of this antibody and recieved the following answer:

"The ChIP was made with the equivalent of 1.5 x 106 cells, this is approximately 10 to 15 ug of chromatin per 10ul antibody."

I hope this is a good starting point for the optimization of your experiments.

Read More

Question
Answer

Ab47915 antibody is sold as whole antiserum. Unpurified antibodies, such as those sold as whole antiserum, ascites or tissue culture supernatant will not have a concentration stated on the datasheet. Antibody concentration is usually determined by protein assay, and serum / ascites / tissue culture supernatant will contain a lot of other proteins, which means the antibody quantification would not be accurate.

I can confirm that for whole antiserum, concentration of antibody is known to very between 1 - 10 mg/ml.

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