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

Overview

  • Product name
    Anti-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
  • Host species
    Mouse
  • Specificity
    By peptide ELISA ab1220 recognizes 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 applications
    Suitable 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, Common marmoset, Rice, Other species
    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 (Cysteine residue). Clones were positively screened by ELISA against the immunising peptide. Clones were negatively screened against both the non-modified equivalent peptide and against a dimethylated K27 peptide.
    (Peptide available as ab1772)

  • Positive control
    • IHC-P: Human kidney tissue; Rat liver tissue. ICC/IF: HeLa cells. Flow Cytometry: Mouse embryonic stem cells. WB: HeLa whole cell lysate.
  • General notes

    ChIP protocols:
    ChIP protocol for cross-linking ChIP (X-ChIP)
    Native ChIP protocol
    Chromatin preparation from tissues for ChIP
    ChIP troubleshooting
    ChIP tips and tricks guide

    This antibody clone [mAbcam 1220] is manufactured by Abcam.

    If you require this antibody in a particular buffer formulation or a particular conjugate for your experiments, please contact orders@abcam.com or you can find further information here.

    Abcam recommended secondaries - Goat Anti-Mouse HRP (ab205719) and Goat Anti-Mouse Alexa Fluor® 488 (ab150113).

Properties

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

  • 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

  • Alarcon V et al investigates the effects of pargyline due to inhibition of LSD1. Levels of LSD1 protein were reduced using siRNA (siLSD1).  Cells were treated with and without siLSD1 and incubated for 24 hours before transfection of HBV genome (A). Western blot analysis shows the reduction of LSD1 after treatment. Covalent post-translational modifications on Histone H3 was determined by ChIP using ab1220 as one of the specific antibodies (C-G). HBV cccDNA and cytoplasmic levels were determined by qPCR (H).

  • 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
    All lanes : 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 Histone H3 (di methyl K9) in MEFs SV40 transformed by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with PFA, permeabilized with 0.2% Triton X-100 in PBS and blocked with 3% BSA for 1 hour at 21°C. Samples were incubated with primary antibody (1/200) for 1 hour at 21°C. A Cy3® conjugated Goat anti-mouse was used as the secondary antibody.

    See Abreview

  • 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.
     

  • Chen L et al investigates role of Trim 28 (Tripartite motif containing 28) in the epithelial to mesenchymal transition which is implicated in cancer metastasis. ChIP was performed using ab1220. ChIP assay was performed in Trim 28 knockdown A549 cells and control (A). ChIP assay was performed in control and Trim28 expressed in A549 cells (B).

  • ChIP was performed using ab1220. Naïve Mouse ES cells were resuspended in PBS and crosslinked with 1% formaldehyde for 5 minutes at room temperature. Crosslinking was quenched with 125m M glycine. A mouse anti IgG (ab18413) was used as a control. ChIP -seq for H3K9me2 in ES cells treated with and without vitamin C (image a). H3K9me2 signal genome-wide was plotted comparing untreated and vitamin C-treated cells (image b). Fold change for average H3K9me2 signal at gene promoters. Most gene promoters display a reduction in H3K9me2 in vitamin C-treated ES cells (image c). ChIP-qPCR for H3K9me2 in ES cells with or without vitamin C at gene promoters. ChIP for IgG was performed as a negative control (image d). Asterisks represent P < 0.05 by t test

  • Anti-Histone H3 (di methyl K9) antibody [mAbcam 1220] - ChIP Grade (ab1220) at 1/1000 dilution + HEK293 at 30 µg

    Secondary
    HRP conjugated polyclonal Sheep anti-Mouse IgG at 1/5000 dilution

    Performed under reducing conditions.

    Predicted band size: 17 kDa



    Blocking buffer: 5% milk

    See Abreview

  • Immunocytochemistry/Immunofluorescence analysis of 1 cell mouse embryo labeling Histone H3 with ab1220 at a dilution of 1/50. Cells were fixed with formaldehyde. Blocking was performed wtih 5% serum for 30 minutes at 37°C. An Alexa Fluor®488 conjugated monoclonal mouse IgG was used as the secondary antibody.

    See Abreview

  • 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.

  • 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 Histone H3 (di methyl K9) in Mouse Tissue sections (Liver, P5) by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with paraformaldehyde and blocked with 5% BSA for 1 hour at 25°C; antigen retrieval was by heat mediation in a citrate buffer. Samples were incubated with primary antibody (1/100 in PBS/Triton 0,05%) for 3 hours at 25°C. An Alexa Fluor® 488 conjugated Goat anti-mouse (1/250) was used as the secondary antibody.

    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
    All lanes : 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

    See Abreview

  • 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

References

This product has been referenced in:
  • He L  et al. A naturally occurring epiallele associates with leaf senescence and local climate adaptation in Arabidopsis accessions. Nat Commun 9:460 (2018). Read more (PubMed: 29386641) »
  • Gao Y & Ge W The histone methyltransferase DOT1L inhibits osteoclastogenesis and protects against osteoporosis. Cell Death Dis 9:33 (2018). Read more (PubMed: 29348610) »
See all 520 Publications for this product

Customer reviews and Q&As

1-10 of 68 Abreviews or Q&A

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

Abcam user community

Verified customer

Submitted Apr 29 2013

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Human Cell lysate - nuclear (B Cells)
Gel Running Conditions
Reduced Denaturing (4-20%)
Loading amount
20 µg
Specification
B Cells
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 Sep 05 2018

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Human Cell lysate - whole cell (HEK293)
Gel Running Conditions
Reduced Denaturing (4-12% Bis-Tris)
Loading amount
30 µg
Specification
HEK293
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 Sep 23 2017

Application
Western blot
Sample
Mouse Cell lysate - nuclear (Hippocampal Primary Cells)
Gel Running Conditions
Reduced Denaturing (15% Gel)
Loading amount
5 µg
Specification
Hippocampal Primary Cells
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 Mar 28 2017

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (adipose stem cells)
Permeabilization
Yes - Triton 0.25%
Specification
adipose stem cells
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
Fixative
Paraformaldehyde

Abcam user community

Verified customer

Submitted Mar 07 2017

Application
Immunocytochemistry/ Immunofluorescence
Sample
Mouse Cell (embryo)
Specification
embryo
Blocking step
Serum as blocking agent for 30 minute(s) · Concentration: 5% · Temperature: 37°C
Fixative
Formaldehyde

Dr. Maki Asami

Verified customer

Submitted Dec 21 2016

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

Abcam user community

Verified customer

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

Abcam user community

Verified customer

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

Abcam user community

Verified customer

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

Mr. Chanhee Jo

Verified customer

Submitted Dec 25 2013

1-10 of 68 Abreviews or Q&A

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