Overview

  • Product name
    Anti-Histone H3 (mono methyl K9) antibody - ChIP Grade
    See all Histone H3 primary antibodies
  • Description
    Rabbit polyclonal to Histone H3 (mono methyl K9) - ChIP Grade
  • Host species
    Rabbit
  • Specificity
    Weak cross reactivity is observed with mono methyl K27 Histone H3. No cross-reactivity is seen with di or tri methyl K27.
  • Tested applications
    Suitable for: IP, WB, IHC-P, Flow Cyt, ChIP, ICC/IFmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Cow, Human, Xenopus laevis, Arabidopsis thaliana, Indian muntjac, Schizosaccharomyces pombe
    Predicted to work with: Mammals
  • Immunogen

    Synthetic peptide within Human Histone H3 aa 1-100 (mono methyl K9) conjugated to Keyhole Limpet Haemocyanin (KLH). The exact sequence is proprietary.
    (Peptide available as ab1771)

  • Positive control
    • Calf Thymus Histone Preparation; Hela whole cell extract

Properties

Applications

Our Abpromise guarantee covers the use of ab9045 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
IP Use at an assay dependent concentration.
WB 1/1000. Detects a band of approximately 15 kDa (predicted molecular weight: 17 kDa). Can be blocked with Histone H3 peptide - mono methyl K9 (ab1771).
IHC-P Use at an assay dependent concentration.
Flow Cyt 1/100.

ab171870 - Rabbit polyclonal IgG, is suitable for use as an isotype control with this antibody.

ChIP Use 4-5µg for 106 cells.
ICC/IF Use at an assay dependent concentration.

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

  • 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 ab9045 (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.

  • Anti-mono methyl lysine 9 of histone H3 (green) has a distribution often associated with euchromatic probes (small foci).  Most of these foci localize to regions that contain obvious enrichments of DNA with DAPI staining (red).  The perinucleolar chromatin is typically a site enriched in monomethylated lysine 9. 

    Top left: Mono-methyl Lys 9 (ab9045); Bottom left: DAPI; Top right: Merge of ab9045 (green) and DAPI (red).

  • ab9045 staining rat liver tissue sections by IHC-P.  Sections were formaldehyde fixed and subjected to heat mediated antigen retrieval in citrate buffer pH 6.0 prior to blocking with 5% serum for 30 minutes at 20°C.  The primary antibody was diluted 1/400 and incubated with the sample for 45 minutes at 20°C.  A HRP-conjugated goat anti-rabbit antibody was used as the secondary.

    See Abreview

  • All lanes : Anti-Histone H3 (mono methyl K9) antibody - ChIP Grade (ab9045) at 1 µg/ml

    Lane 2 : Human Histone H3 (unmodified ) peptide (ab7228)
    Lane 3 : Human Histone H3 (mono methyl K27) peptide (ab1780)
    Lane 4 : Human Histone H3 (di methyl K27) peptide (ab1781)
    Lane 5 : Human Histone H3 (tri methyl K27) peptide (ab1782)
    Lane 6 : Human Histone H3 (mono methyl K4) peptide (ab1340)
    Lane 7 : Human Histone H3 (mono methyl K9) peptide (ab1771)

    Predicted band size: 17 kDa



    Rabbit polyclonal to Histone H3 K9 Methyl K9 (1/1000)

    Peptides at 1 ug/ml

    1XTBS, 5%BSA, 0.5% Tween

    This antibody shows significantly greater reactivity with mono methyl K9. This can be seen in lane 7, as the addition of ab1771 (mono methyl K9) completely blocks the activity of ab9045. Weaker cross-reactivity is seen against mono methyl K27. This is shown in lane 3, as the addition of ab1780 only partially blocks the activity of ab9045.

  • All lanes : Anti-Histone H3 (mono methyl K9) antibody - ChIP Grade (ab9045) at 1 µg/ml

    Lane 1 : Calf Thymus Histone Preparation Nuclear Lysate (ab121)
    Lane 2 : Calf Thymus Histone Preparation Nuclear Lysate (ab121) with Human Histone H3 (unmodified ) peptide (ab7228) at 0.5 µg/ml
    Lane 3 : Calf Thymus Histone Preparation Nuclear Lysate (ab121) with Human Histone H3 (mono methyl K4) peptide (ab1340) at 0.5 µg/ml
    Lane 4 : Calf Thymus Histone Preparation Nuclear Lysate (ab121) with Human Histone H3 (di methyl K4) peptide (ab7768) at 0.5 µg/ml
    Lane 5 : Calf Thymus Histone Preparation Nuclear Lysate (ab121) with Human Histone H3 (tri methyl K4) peptide (ab1342) at 0.5 µg/ml
    Lane 6 : Calf Thymus Histone Preparation Nuclear Lysate (ab121) with Human Histone H3 (mono methyl K9) peptide (ab1771) at 0.5 µg/ml
    Lane 7 : Calf Thymus Histone Preparation Nuclear Lysate (ab121) with Human Histone H3 (di methyl K9) peptide (ab1772) at 0.5 µg/ml
    Lane 8 : Calf Thymus Histone Preparation Nuclear Lysate (ab121) with Human Histone H3 (tri methyl K9) peptide (ab1773) at 0.5 µg/ml
    Lane 9 : Calf Thymus Histone Preparation Nuclear Lysate (ab121) with Human Histone H3 (mono methyl K27) peptide (ab1780) at 0.5 µg/ml
    Lane 10 : Calf Thymus Histone Preparation Nuclear Lysate (ab121) with Human Histone H3 (di methyl K27) peptide (ab1781) at 0.5 µg/ml
    Lane 11 : Calf Thymus Histone Preparation Nuclear Lysate (ab121) with Human Histone H3 (tri methyl K27) peptide (ab1782) at 0.5 µg/ml

    Lysates/proteins at 0.5 µg per lane.

    Secondary
    All lanes : IRDye 680 Conjugated Goat Anti-Rabbit IgG (H+L) at 1/10000 dilution

    Performed under reducing conditions.

    Predicted band size: 17 kDa

  • Histone H3 (mono methyl K9) was immunoprecipitated using 0.5mg Hela whole cell extract, 5µg of Rabbit polyclonal to Histone H3 (mono methyl K9) and 50µl of protein G magnetic beads (+). No antibody was added to the control (-).
    The antibody was incubated under agitation with Protein G beads for 10min, Hela whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.
    Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab9045.
    Secondary: Anti-rabbit IgG VeriBlot for IP secondary antibody (HRP) (ab131366) at 1/1000 dilution.
    Band: 17kDa: Histone H3 (mono methyl K9).
  • ICC/IF image of ab9045 stained HeLa cells. The cells were 100% methanol fixed (5 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab9045, 1µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 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 1.43µM. This antibody also gave a positive result in 100% methanol fixed (5 min) HepG2, Hek293 and MCF7 cells at 1µg/ml, and in 4% PFA fixed (10 min) HeLa, Hek293, HepG2 amd MCF7 cells at 1µg/ml.

References

This product has been referenced in:
  • Maia LL  et al. JMJD1A, H3K9me1, H3K9me2 and ADM expression as prognostic markers in oral and oropharyngeal squamous cell carcinoma. PLoS One 13:e0194884 (2018). Read more (PubMed: 29590186) »
  • Lyu G  et al. TGF-ß signaling alters H4K20me3 status via miR-29 and contributes to cellular senescence and cardiac aging. Nat Commun 9:2560 (2018). Read more (PubMed: 29967491) »
See all 70 Publications for this product

Customer reviews and Q&As

1-10 of 19 Abreviews or Q&A

Abcam has not validated the combination of species/application used in this Abreview.
Application
Western blot
Sample
Saccharomyces cerevisiae Cell lysate - whole cell (Whole cell lysate)
Gel Running Conditions
Reduced Denaturing (15%)
Loading amount
60000 cells
Specification
Whole cell lysate
Blocking step
Odyssey blocking buffer as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 50% · Temperature: 25°C

Abcam user community

Verified customer

Submitted Aug 08 2016

Application
Immunohistochemistry (Frozen sections)
Sample
Mouse Tissue sections (Skin)
Permeabilization
No
Specification
Skin
Blocking step
Serum as blocking agent for 30 minute(s) · Concentration: 10% · Temperature: 22°C
Fixative
Paraformaldehyde

Ahmar Aziz

Verified customer

Submitted Jul 08 2016

Application
Immunocytochemistry/ Immunofluorescence
Sample
Mouse Cell (Brain cultured cells)
Specification
Brain cultured cells
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: RT°C
Fixative
Paraformaldehyde

Abcam user community

Verified customer

Submitted Oct 12 2015

Application
Immunohistochemistry (PFA perfusion fixed frozen sections)
Sample
Mouse Tissue sections (Brain)
Permeabilization
Yes - Triton x-100, 0.01%
Specification
Brain
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: RT°C
Fixative
Paraformaldehyde

Abcam user community

Verified customer

Submitted Jun 18 2015

Application
Immunohistochemistry (Frozen sections)
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: RT°C
Sample
Mouse Tissue sections (Brain)
Specification
Brain
Permeabilization
Yes - Triton x-100, 0.01%
Fixative
Paraformaldehyde

Abcam user community

Verified customer

Submitted Mar 30 2015

Application
Western blot
Loading amount
40 µg
Gel Running Conditions
Reduced Denaturing (10% PAGE)
Sample
Mouse Cell lysate - whole cell (Cardiomyocytes)
Specification
Cardiomyocytes
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C

Abcam user community

Verified customer

Submitted Jan 30 2015

Abcam has not validated the combination of species/application used in this Abreview.
Application
Immunocytochemistry/ Immunofluorescence
Sample
Cyanidioschyzon merolae Cell (Synchronous culture using light/dark cycle)
Specification
Synchronous culture using light/dark cycle
Fixative
Paraformaldehyde
Blocking step
BSA as blocking agent for 30 minute(s) · Concentration: 5% · Temperature: 37°C

俊之 曾根

Verified customer

Submitted Feb 12 2013

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Human Cell lysate - nuclear (rhabdomyosarcoma cell lines)
Loading amount
25 µg
Specification
rhabdomyosarcoma cell lines
Gel Running Conditions
Reduced Denaturing (10-20% tris glycine)
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 23°C

Abcam user community

Verified customer

Submitted Jan 30 2013

Answer

Below I have summarized our conversation and answers to your questions.

For detecting nuclear proteins via western blot we recommend using a RIPA buffer or cell fractionation protocol in preparation of your cell lysate. I attached our protocol book to this email for information regarding specific cell lysate preparation procedures. Below are the fractionation kits that we talked about during your phone call:
Nuclear Extraction Kit (ab113474) https://www.abcam.com/episeeker-nuclear-extraction-kit-ab113474.html
Histone Extraction Kit (ab113476)
https://www.abcam.com/episeeker-histone-extraction-kit-ab113476.html

The Histone Extraction Kit can be used to extract histones from as low as 1 million cells or 1 mg of tissue as stated on the datasheet. These lower limits are good guidelines for the Nuclear Extraction Kit as well.

Since ab1220 (anti-histone H3 di methyl K9) is the only antibody in your provided list that is raised in mouse (the others are raised in rabbit), we discussed substituting the rabbit monoclonal antibody ab32521 (https://www.abcam.com/histone-h3-di-methyl-k9-antibody-y49-ab32521.html) (anti-histone H3 di methyl K9) so you can use the same secondary for each antibody. A list of our anti-rabbit secondary antibodies is given through the link below, depending on the detection method and conjugation that you prefer:
https://www.abcam.com/search?AppliedFacets.FacetProductType=Secondary+antibodies&AppliedFacets.FacetReactivity=Rabbit&IsFacetsOnly=False&PageNumber=1&Keywords[0]=rabbit&X-Requested-With=XMLHttpRequest

Together these antibodies could be used to detect the presence of histone H3 in your samples as well as the methylation status of Lysine 9. To complete the spectrum, Abcam also sells an anti-histone H3 mono methyl K9 antibody (ab9045, https://www.abcam.com/histone-h3-mono-methyl-k9-antibody-chip-grade-ab9045.html). As we discussed, ab3790 (anti-KMT8/Riz1/Riz2) detects the presence of PR domain zinc finger protein 2, which is a histone methyltransferase that specifically methylates Lysine 9 of histone H3 according to SwissProt (http://www.uniprot.org/uniprot/Q13029), thus explaining its connection to the anti-histone H3 antibodies that detect the methylation status of Lysine 9

Read More
Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunocytochemistry/ Immunofluorescence
Blocking step
Serum as blocking agent for 30 minute(s) · Concentration: 3% · Temperature: 37°C
Sample
Mouse Cell (Zygote)
Specification
Zygote
Permeabilization
Yes - 0.25% ~0.5% Triton
Fixative
Formaldehyde

Dr. Maki Asami

Verified customer

Submitted Oct 30 2012

1-10 of 19 Abreviews or Q&A

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

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