Anti-Histone H3 (di methyl K9) antibody [mAbcam 1220] - ChIP Grade (ab1220)
Key features and details
- Mouse monoclonal [mAbcam 1220] to Histone H3 (di methyl K9) - ChIP Grade
- Suitable for: ICC/IF, WB, ELISA, IHC-P, ChIP
- Reacts with: Cow, Human, Arabidopsis thaliana, Drosophila melanogaster, Rice
- Isotype: IgG2a
Related conjugates and formulations
Overview
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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, WB, ELISA, IHC-P, ChIPmore details -
Species reactivity
Reacts with: Cow, Human, Arabidopsis thaliana, Drosophila melanogaster, Rice
Predicted to work with: Mouse, Rat, Sheep, Chicken, Saccharomyces cerevisiae, Xenopus laevis, Caenorhabditis elegans, Schizosaccharomyces pombe, Corn, Common marmoset, Other species -
Immunogen
Synthetic peptide. This information is proprietary to Abcam and/or its suppliers.
(Peptide available asab1772) -
Positive control
- IHC-P: Human kidney tissue; Mouse liver tissue. WB: HeLa whole cell lysate; Calf thymus histone lysate; HEK-293 lysate. ChIP: AFM ChIP primer pair ab269259; Chromatin prepared from U2OS cells. ICC/IF: HeLa cells.
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General notes
Learn about ChIP assay kits, other ChIP antibodies, and more in the ChIP assay guide.
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 guideThis 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.
The Life Science industry has been in the grips of a reproducibility crisis for a number of years. Abcam is leading the way in addressing this with our range of recombinant monoclonal antibodies and knockout edited cell lines for gold-standard validation. Please check that this product meets your needs before purchasing.
If you have any questions, special requirements or concerns, please send us an inquiry and/or contact our Support team ahead of purchase. Recommended alternatives for this product can be found below, along with publications, customer reviews and Q&As
Properties
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Form
Liquid -
Storage instructions
Shipped 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 buffer
pH: 7.40
Preservative: 0.02% Sodium azide
Constituents: PBS, 6.97% L-Arginine -
Concentration information loading...
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Purity
Protein G purified -
Clonality
Monoclonal -
Clone number
mAbcam 1220 -
Isotype
IgG2a -
Light chain type
kappa -
Research areas
Associated products
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Alternative Versions
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ChIP Related Products
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Compatible Secondaries
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Corresponding Unmodified Peptide
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Immunizing Peptide (Blocking)
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Isotype control
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Positive Controls
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Recombinant Protein
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Related Products
- Histone H3 (K9) Methyltransferase Activity Quantification Assay Kit (ab113453)
- Histone H3 (di-methyl K9) Quantification Kit (Colorimetric) (ab115062)
- Prestained Protein Ladder - Broad molecular weight (10 - 245 kDa) (ab116028)
- Human Histone H3 (tri methyl K4) peptide (ab1342)
- Human Histone H3 (tri methyl K9) peptide (ab1773)
- Human Histone H3 (tri methyl K27) peptide (ab1782)
- Human Histone H3 (unmodified) peptide (ab7228)
Applications
The Abpromise guarantee
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 |
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ICC/IF | (16) |
Use a concentration of 5 µg/ml.
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WB | (33) |
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).
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ELISA |
Use a concentration of 0.00025 - 1 µg/ml.
when testing with immunogen peptide. |
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IHC-P | (7) |
Use at an assay dependent concentration.
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ChIP | (11) |
Use 2-4 µg for 25 µg of chromatin.
Use AFM ChIP primer pair ab269259 as positive control. |
Notes |
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ICC/IF
Use a concentration of 5 µg/ml. |
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). |
ELISA
Use a concentration of 0.00025 - 1 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. Use AFM ChIP primer pair ab269259 as positive control. |
Target
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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
modificationsAcetylation 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
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Database links
- Entrez Gene: 8350 Human
- Entrez Gene: 8351 Human
- Entrez Gene: 8352 Human
- Entrez Gene: 8353 Human
- Entrez Gene: 8354 Human
- Entrez Gene: 8355 Human
- Entrez Gene: 8356 Human
- Entrez Gene: 8357 Human
see all -
Alternative names
- H3 histone family member E pseudogene antibody
- H3 histone family, member A antibody
- H3/A antibody
see all
Images
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ChIP - Anti-Histone H3 (di methyl K9) antibody [mAbcam 1220] - ChIP Grade (ab1220)Image from Alarcon V et al., Sci Rep 13(6), Fig 4. Doi: 10.1038/srep25901. Reproduced under the Creative Commons license http://creativecommons.org/licenses/by/4.0/
Alarcon V et al investigates the effects of pargyline due to inhibition of LSD1. Levels of LSD1 protein were reduced using siRNA (siLSD1). Huh7 (Human hepatocellular carcinoma) 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).
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Immunocytochemistry/ Immunofluorescence - Anti-Histone H3 (di methyl K9) antibody [mAbcam 1220] - ChIP Grade (ab1220)
ab1220 staining Histone H3 (di methyl K9) in HeLa (Human cervix adenocarcinoma epithelial cell) cells. The cells were fixed with 4% PFA (10 mins), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated overnight at +4°C with ab1220 at 5 µg/ml and ab190573, Rabbit monoclonal to alpha Tubulin (Alexa Fluor® 647), at 2 µg/ml (shown in red). The secondary antibody (shown in green) was ab150117, Alexa Fluor® 488 Goat anti-Mouse IgG (H+L) used at a 1/1000 dilution for 1h at room temperature. Nuclear DNA was labelled with DAPI (shown in blue).
Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown.
This product also gave a positive outcome under the same testing conditions in HeLa cells fixed with 100% methanol (5 mins). -
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 withHuman Histone H3 (di methyl K9) peptide (ab1772) at 1 µg/ml
Lane 5 : Calf thymus histone lysate withHuman 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 withHuman 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 -
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.
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ChIP - Anti-Histone H3 (di methyl K9) antibody [mAbcam 1220] - ChIP Grade (ab1220)Image from Chen L et al., PLoS One 9(7), Fig 6. Doi: 10.1371/journal.pone.0101040 Reproduced under the Creative Commons license http://creativecommons.org/licenses/by/4.0/
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).
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ChIP - Anti-Histone H3 (di methyl K9) antibody [mAbcam 1220] - ChIP Grade (ab1220)Image from Ebata KT et al., Epigenetics Chromatin 10(36), Fig 2. Doi: 10.1186/s13072-017-0143-3 Reproduced under the Creative Commons license http://creativecommons.org/licenses/by/4.0/.
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
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Western blot - Anti-Histone H3 (di methyl K9) antibody [mAbcam 1220] - ChIP Grade (ab1220)This image is courtesy of an anonymous abreview.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 kDaBlocking buffer: 5% milk
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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H3 (di methyl K9) antibody [mAbcam 1220] - ChIP Grade (ab1220)
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.
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ELISA using ab1220 at varying antibody concentrations.
The purple line indicates binding to the Human Histone H3 (di methyl K9) peptide ab1772. Binding to the following peptides was not seen:
Human Histone H3 (unmodified ) peptide (ab2903),
Human Histone H3 (mono methyl K9) peptide (ab1771),
Human Histone H3 (tri methyl K9) peptide (ab1773),
Human Histone H3 (di methyl K27) peptide (ab1781),
Human Histone H3 (tri methyl K27) peptide (ab1782),
Human Histone H3 (mono methyl K4) peptide (ab1340),
Human Histone H3 (di methyl K4) peptide (ab7768),
Human Histone H3 (tri methyl K4) peptide (ab1342).This indicates the specificity of ab1220 for di methyl K9 of Histone H3.
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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 minutesLane 1 : Marker.
All blocking and antibody incubation steps were done with 5% milk in 20mM Tris-HCL, and 0.1% TWEEN-20.
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Western blot - Anti-Histone H3 (di methyl K9) antibody [mAbcam 1220] - ChIP Grade (ab1220)This image is courtesy of an anonymous AbreviewAll 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
Datasheets and documents
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SDS download
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Datasheet download
References (886)
ab1220 has been referenced in 886 publications.
- Wang S et al. Inheritance of paternal DNA damage by histone-mediated repair restriction. Nature 613:365-374 (2023). PubMed: 36544019
- Li W et al. 4,5-Dimethoxycanthin-6-one is a novel LSD1 inhibitor that inhibits proliferation of glioblastoma cells and induces apoptosis and pyroptosis. Cancer Cell Int 22:32 (2022). PubMed: 35042538
- Anderson EM et al. The histone methyltransferase G9a mediates stress-regulated alcohol drinking. Addict Biol 27:e13060 (2022). PubMed: 34013595
- Finke D et al. Histone deacetylase 4 deletion broadly affects cardiac epigenetic repression and regulates transcriptional susceptibility via H3K9 methylation. J Mol Cell Cardiol 162:119-129 (2022). PubMed: 34492228
- Yang S et al. Solanaceous plants switch to cytokinin-mediated immunity against Ralstonia solanacearum under high temperature and high humidity. Plant Cell Environ 45:459-478 (2022). PubMed: 34778967