Anti-Histone H3 (di methyl K9) antibody [mAbcam 1220] - ChIP Grade (ab1220)

Overview

  • Product nameAnti-Histone H3 (di methyl K9) antibody [mAbcam 1220] - ChIP Grade
    See all Histone H3 primary antibodies
  • Description
    Mouse monoclonal [mAbcam 1220] to Histone H3 (di methyl K9) - ChIP Grade
  • SpecificityBy peptide ELISA ab1220 recognises di methyl K9, but not unmodified K9, mono methyl K9, tri methyl K9, di methyl K27, tri methyl K27, mono methyl K4, di methyl K4 or tri methyl K4. By Western blot ab1220 is blocked by di methyl K9, but not by unmodified K9, mono methyl K9, tri methyl K9, di methyl K27, tri methyl K27, mono methyl K4, di methyl K4 or tri methyl K4. This indicates the specificity of ab1220 for di methyl K9 of Histone H3.
  • Tested applicationsSuitable for: ICC/IF, IP, WB, Flow Cyt, IHC-Fr, ELISA, IHC-P, ChIPmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Chicken, Cow, Human, Xenopus laevis, Arabidopsis thaliana, Caenorhabditis elegans, Drosophila melanogaster, Schizosaccharomyces pombe, Corn, Marmoset (common), Rice, Other
    Predicted to work with: Sheep, Saccharomyces cerevisiae
  • Immunogen

    Synthetic peptide corresponding to Human Histone H3 aa 1-100 (di methyl K9) conjugated to Keyhole Limpet Haemocyanin (KLH) (Cysteine residue).
    (Peptide available as ab1772)

  • General notes

    Hybridomas were prepared and the resulting clones were positively screened by ELISA against the immunising peptide conjugated to BSA. Clones were negatively screened against both the non-modified equivalent peptide and against a peptide corresponding to dimethylated K27.

    ab170191 - Mouse monoclonal IgG2a, is suitable for use as an isotype control with this antibody.

    Alternative versions available:
    Anti-Histone H3 (di methyl K9) antibody (Alexa Fluor® 488) [mAbcam 1220] - (ab203850)
    Anti-Histone H3 (di methyl K9) antibody (Alexa Fluor® 647) [mAbcam 1220] - (ab203851)
    Anti-Histone H3 (di methyl K9) antibody (HRP) [mAbcam 1220] - (ab62168)

Properties

  • FormLiquid
  • Storage instructionsShipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C or -80°C. Avoid freeze / thaw cycle.
  • Storage bufferpH: 7.50
    Preservative: 0.02% Sodium azide
    Constituent: PBS

    Some batches of this product contain 0.4M Arginine as a stabilising agent. If you would like information about the formulation of a specific lot, please contact our scientific support team who will be happy to help.
  • Concentration information loading...
  • PurityIgG fraction
  • ClonalityMonoclonal
  • Clone numbermAbcam 1220
  • IsotypeIgG2a
  • Light chain typekappa
  • Research areas

Applications

Our Abpromise guarantee covers the use of ab1220 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 Use a concentration of 5 µg/ml.
IP Use at an assay dependent concentration.
WB Use a concentration of 1 - 5 µg/ml. Detects a band of approximately 17 kDa (predicted molecular weight: 17 kDa).Can be blocked with Human Histone H3 (di methyl K9) peptide (ab1772).
Flow Cyt Use at an assay dependent concentration.

ab170191 - Mouse monoclonal IgG2a, is suitable for use as an isotype control with this antibody.

IHC-Fr Use at an assay dependent concentration.
ELISA Use a concentration of 1 - 0.00025 µg/ml. when testing with immunogen peptide.
IHC-P Use at an assay dependent concentration.
ChIP Use 2-4 µg for 25 µg of chromatin.

Target

  • FunctionCore 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 similaritiesBelongs to the histone H3 family.
  • Developmental stageExpressed 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 localizationNucleus. Chromosome.
  • Information by UniProt
  • Database links
    see all
  • Alternative names
    • HIST1 cluster, H3J antibody
    • Histone gene cluster 1, H3 histone family, member E antibody
    • Histone gene cluster 1, H3G antibody
    • H3 histone family, member J antibody
    • HIST1 cluster, H3E antibody
    • HIST1 cluster, H3I antibody
    • Histone gene cluster 1, H3C antibody
    • FLJ92264 antibody
    • H3 histone family, member A antibody
    • H3 histone family, member B antibody
    • H3 histone family, member C antibody
    • H3 histone family, member D antibody
    • H3 histone family, member F antibody
    • H3 histone family, member H antibody
    • H3 histone family, member I antibody
    • H3 histone family, member K antibody
    • H3 histone family, member L antibody
    • H3 histone family, member T antibody
    • H3 histone, family 3A antibody
    • H3.1 antibody
    • H3.3A antibody
    • H3/a antibody
    • H3/b antibody
    • H3/c antibody
    • H3/d antibody
    • h3/f antibody
    • H3/h antibody
    • H3/i antibody
    • H3/j antibody
    • H3/k antibody
    • H3/l antibody
    • H3/t antibody
    • H31_HUMAN antibody
    • H3F1K antibody
    • H3F3 antibody
    • H3F3A antibody
    • H3FA antibody
    • H3FB antibody
    • H3FC antibody
    • H3FD antibody
    • H3FF antibody
    • H3FH antibody
    • H3FI antibody
    • H3FJ antibody
    • H3FK antibody
    • H3FL antibody
    • HIST1 cluster, H3A antibody
    • HIST1 cluster, H3B antibody
    • HIST1 cluster, H3C antibody
    • HIST1 cluster, H3D antibody
    • HIST1 cluster, H3F antibody
    • HIST1 cluster, H3G antibody
    • HIST1 cluster, H3H 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 1, H3b antibody
    • Histone 1, H3c antibody
    • Histone 1, H3d antibody
    • Histone 1, H3e antibody
    • Histone 1, H3f antibody
    • Histone 1, H3g antibody
    • Histone 1, H3h antibody
    • Histone 1, H3i antibody
    • Histone 1, H3j antibody
    • histone 3, H3 antibody
    • histone cluster 1 H3 family member a antibody
    • histone cluster 1 H3 family member b antibody
    • histone cluster 1 H3 family member c antibody
    • histone cluster 1 H3 family member d antibody
    • histone cluster 1 H3 family member e antibody
    • histone cluster 1 H3 family member f antibody
    • histone cluster 1 H3 family member g antibody
    • histone cluster 1 H3 family member h antibody
    • histone cluster 1 H3 family member i antibody
    • histone cluster 1 H3 family member j antibody
    • Histone cluster 1, H3a antibody
    • Histone cluster 1, H3b antibody
    • Histone cluster 1, H3c antibody
    • Histone cluster 1, H3d antibody
    • Histone cluster 1, H3e antibody
    • Histone cluster 1, H3f antibody
    • Histone cluster 1, H3g antibody
    • Histone cluster 1, H3i antibody
    • Histone cluster 1, H3j antibody
    • Histone gene cluster 1, H3 histone family, member A antibody
    • Histone gene cluster 1, H3 histone family, member B antibody
    • Histone gene cluster 1, H3 histone family, member C antibody
    • Histone gene cluster 1, H3 histone family, member D antibody
    • Histone gene cluster 1, H3 histone family, member F antibody
    • Histone gene cluster 1, H3 histone family, member G antibody
    • Histone gene cluster 1, H3 histone family, member H antibody
    • Histone gene cluster 1, H3 histone family, member I antibody
    • Histone gene cluster 1, H3 histone family, member J antibody
    • Histone gene cluster 1, H3A antibody
    • Histone gene cluster 1, H3B antibody
    • Histone gene cluster 1, H3D antibody
    • Histone gene cluster 1, H3E antibody
    • Histone gene cluster 1, H3F antibody
    • Histone gene cluster 1, H3H antibody
    • Histone gene cluster 1, H3I antibody
    • Histone gene cluster 1, H3J antibody
    • Histone H 3 antibody
    • Histone H3.1 antibody
    • histone H3.1t antibody
    • Histone H3.2 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
    • Histone H3/m antibody
    • Histone H3/o antibody
    see all

Anti-Histone H3 (di methyl K9) antibody [mAbcam 1220] - ChIP Grade images

  • Chromatin was prepared from U2OS cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10min. The  ChIP was performed with 25µg of chromatin, 2µg of  ab1220 (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.
     

  • All lanes : Anti-Histone H3 (di methyl K9) antibody [mAbcam 1220] - ChIP Grade (ab1220)

    Lane 1 : Calf thymus histone lysate
    Lane 2 : Calf thymus histone lysate with Histone H3 peptide - unmodified at 1 µg/ml
    Lane 3 : Calf thymus histone lysate with Human Histone H3 (mono methyl K9) peptide (ab1771) at 1 µg/ml
    Lane 4 : Calf thymus histone lysate with Human Histone H3 (di methyl K9) peptide (ab1772) at 1 µg/ml
    Lane 5 : Calf thymus histone lysate with Human Histone H3 (tri methyl K9) peptide (ab1773) at 1 µg/ml
    Lane 6 : Calf thymus histone lysate with Human Histone H3 (di methyl K4) peptide (ab7768) at 1 µg/ml
    Lane 7 : Calf thymus histone lysate with Human Histone H3 (di methyl K27) peptide (ab1781) at 1 µg/ml

    Secondary
    Rabbit Anti-Mouse IgG H&L (HRP) (ab6728) at 1/5000 dilution
    Developed using the ECL technique

    Performed under reducing conditions.

    Predicted band size : 17 kDa


    Exposure time : 1 minute
  • ab1220 staining human kidney sections by IHC-P using EXPOSE IHC detection kit (ab80436). Formalin fixed paraffin embedded tissue sections were pre-treated using heat mediated antigen retrieval (using a pressure cooker) with sodium citrate buffer (pH6) for 30 mins. The section was incubated with ab1220, 5µg/ml, for 1 hour at room temperature. DAB was used as the chromogen and the section was counterstained with haematoxylin and mounted with DPX.

  • Interphase HeLa cells were fixed in paraformaldehyde and labelled with ab1220 (1/500). The staining is shown in green. The cells were counterstained with DAPI (red).

  • ab1220 staining mouse embryonic stem cells by flow cytometry (gated on all living cells). The cells were trypsinized and stained with the antibody at 1ug/1.5 x 105 cells in a permeabilization buffer. A Cy3® conjugated goat anti-mouse antibody was used as the secondary.

    See Abreview

  • ELISA using ab1220 at varying antibody concentrations.

    The purple line indicates binding to the di methyl K9 peptide ab1772. Binding to the following peptides was not seen:
    unmodified K9 (ab2903),
    mono methyl K9 (ab1771),
    tri methyl K9 (ab1773),
    di methyl K27 (ab1781),
    tri methyl K27 peptide (ab1782),
    mono methyl K4 (ab1340),
    di methyl K4 (ab7768),
    tri methyl K4 (ab1342).

    This indicates the specificity of ab1220 for di methyl K9 of Histone H3.

  • Anti-Histone H3 (di methyl K9) antibody [mAbcam 1220] - ChIP Grade (ab1220) at 1 µg/ml + HeLa (Human epitherlial carcinoma cell line) Whole Cell Lysate at 20 µg

    Secondary
    Goat polyclonal to Mouse IgG-H&L- Pre-Adsorbed (HRP) at 1/10000 dilution
    Developed using the ECL technique

    Performed under reducing conditions.

    Predicted band size : 17 kDa
    Observed band size : 17 kDa


    Exposure time : 5 minutes

    Lane 1 : Marker.

    All blocking and antibody incubation steps were done with 5% milk in 20mM Tris-HCL, and0.1% TWEEN-20.

  • ab1220 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-mouse antibody was used as the secondary.

    See Abreview

  • All lanes : Anti-Histone H3 (di methyl K9) antibody [mAbcam 1220] - ChIP Grade (ab1220) at 2.5 µg/ml

    Lane 1 : Fruit fly embryo tissue lysate - nuclear at 4.5 µg
    Lane 2 : Fruit fly embryo tissue lysate - nuclear at 1.5 µg
    Lane 3 : Fruit fly embryo tissue lysate - nuclear at 0.5 µg

    Secondary
    HRP-conjugated Goat anti-mouse IgG polyclonal at 1/2000 dilution
    Developed using the ECL technique

    Performed under reducing conditions.

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


    Exposure time : 1 minute

    This image is courtesy of an anonymous Abreview

    See Abreview

References for Anti-Histone H3 (di methyl K9) antibody [mAbcam 1220] - ChIP Grade (ab1220)

This product has been referenced in:
  • Kong L  et al. A primary role of TET proteins in establishment and maintenance of De Novo bivalency at CpG islands. Nucleic Acids Res 44:8682-8692 (2016). ChIP ; Human . Read more (PubMed: 27288448) »
  • Martin KA  et al. Epstein-Barr Virus Oncoprotein LMP1 Mediates Epigenetic Changes in Host Gene Expression through PARP1. J Virol 90:8520-30 (2016). Read more (PubMed: 27440880) »

See all 183 Publications for this product

Product Wall

Abcam guarantees this product to work in the species/application used in this Abreview.
Application ChIP
Sample Mouse Cell lysate - whole cell (C2C12 mouse myoblast cells)
Specification C2C12 mouse myoblast cells
Type Cross-linking (X-ChIP)
Duration of cross-linking step: 10 minute(s) and 0 second(s)
Specification of the cross-linking agent: 0.75% formaldehyde
Detection step Real-time PCR
Positive control Control C2C12 cells at day 2 of differentiation
Negative control Beads only and mouse IgG were the negative controls
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Submitted Apr 29 2013

Application Western blot
Sample Horse Cell lysate - whole cell (fibroblasts)
Gel Running Conditions Reduced Denaturing (15%)
Loading amount 200000 cells
Specification fibroblasts
Blocking step Milk as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 4°C
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Submitted Apr 12 2016

Application ChIP
Sample Caenorhabditis elegans Tissue lysate - whole (Whole worm mixed population)
Negative control input
Specification Whole worm mixed population
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: formaldehyde
Positive control samples
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Submitted Sep 15 2015

Application Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Blocking step BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
Antigen retrieval step Heat mediated - Buffer/Enzyme Used: Na-citrate pH6
Sample Mouse Tissue sections (Liver, P5)
Specification Liver, P5
Permeabilization Yes - Triton 0,05%
Fixative Paraformaldehyde
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Submitted Oct 06 2014

Application Western blot
Loading amount 20 µg
Gel Running Conditions Reduced Denaturing (15%)
Sample Human Purified protein (human hepatocarcimona, HepG2)
Specification human hepatocarcimona, HepG2
Treatment Various media condition for 24hrs
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
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Submitted Dec 25 2013

Application Western blot
Loading amount 30 µg
Gel Running Conditions Reduced Denaturing (12)
Sample Human Cell lysate - whole cell (Human Embryonic kidney cell lines)
Specification Human Embryonic kidney cell lines
Blocking step BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 2% · Temperature: 25°C
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Submitted Nov 25 2013

Application Immunocytochemistry/ Immunofluorescence
Blocking step BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: 25°C
Sample Mouse Cell (IMR32, Human Neuroblatoma)
Specification IMR32, Human Neuroblatoma
Permeabilization Yes - 0.3% triton X 100
Fixative Paraformaldehyde
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Submitted Nov 08 2013

Application Western blot
Loading amount 4.5 µg
Gel Running Conditions Reduced Denaturing (12)
Sample Caenorhabditis elegans Tissue lysate - nuclear (C. elegans whole body)
Specification C. elegans whole body
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 23°C
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Submitted Oct 29 2013

Application Western blot
Loading amount 4.5 µg
Gel Running Conditions Reduced Denaturing (12)
Sample Fruit fly (Drosophila melanogaster) Tissue lysate - nuclear (Fruit fly embryo)
Specification Fruit fly embryo
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 23°C
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Submitted Oct 28 2013

Application Western blot
Loading amount 20 µg
Gel Running Conditions Reduced Denaturing (12)
Sample Human Cell lysate - whole cell (Human HeLa cell line)
Specification Human HeLa cell line
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 23°C
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Submitted Oct 28 2013

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