Anti-Histone H3 (tri methyl K4) antibody [mAbcam12209] - ChIP Grade (ab12209)
Key features and details
- Mouse monoclonal [mAbcam12209] to Histone H3 (tri methyl K4) - ChIP Grade
- Suitable for: WB, ICC/IF, ChIP, ELISA, Flow Cyt (Intra)
- Reacts with: Rat, Cow, Human
- Isotype: IgG1
Overview
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Product name
Anti-Histone H3 (tri methyl K4) antibody [mAbcam12209] - ChIP Grade
See all Histone H3 primary antibodies -
Description
Mouse monoclonal [mAbcam12209] to Histone H3 (tri methyl K4) - ChIP Grade -
Host species
Mouse -
Specificity
ab12209 is strongly blocked in Western blotting on histones by tri methyl K4, weakly by di methyl K4 and very weakly by mono methyl K4 peptides. It is not blocked by non-modified peptides. By ELISA the antibody binds to the tri methyl K4 peptide and at high antibody concentrations to di and mono methyl K4 peptides. It does not bind to unmodified, mono, di or tri methyl K9 or di or tri methyl K27 peptides. Not suitable for blocking with milk in Western blot (see Application notes).
Our internal testing has revealed that the supernatant contains a very small fraction of IgG2b. IgG1 remains the main isotype.
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Tested applications
Suitable for: WB, ICC/IF, ChIP, ELISA, Flow Cyt (Intra)more details -
Species reactivity
Reacts with: Rat, Cow, Human
Predicted to work with: Mouse, Saccharomyces cerevisiae, Xenopus laevis, Arabidopsis thaliana, Caenorhabditis elegans, Drosophila melanogaster, Schizosaccharomyces pombe, Zebrafish, Mammals, Neurospora crassa -
Immunogen
Synthetic peptide within Human Histone H3 aa 1-100 (tri methyl K4) conjugated to keyhole limpet haemocyanin. The exact sequence is proprietary.
(Peptide available asab1342) -
Positive control
- Flow Cyt (Intra): HeLa cells; ICC/IF: HeLa cells; WB: Calf Thymus Histone Preparation Nuclear Lysate; ChIP: U2OS cells.
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General notes
This antibody clone is manufactured by Abcam. If you require a custom buffer formulation or conjugation for your experiments, please contact orders@abcam.com.
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
IgG fraction -
Clonality
Monoclonal -
Clone number
mAbcam12209 -
Myeloma
Sp2/0-Ag14 -
Isotype
IgG1 -
Light chain type
kappa -
Research areas
Associated products
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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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Recombinant Protein
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Related Products
Applications
The Abpromise guarantee
Our Abpromise guarantee covers the use of ab12209 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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WB | (2) |
Use a concentration of 1 - 5 µg/ml. Detects a band of approximately 17 kDa (predicted molecular weight: 15 kDa).Can be blocked with Human Histone H3 (tri methyl K4) peptide (ab1342). NOT SUITABLE for blocking with milk. Block in 5% BSA for 1 hour. Our labs have investigated the blocking conditions for this antibody and found that milk significantly decreases the signal and is therefore not a suitable blocking agent for this antibody (see Western Blot image).
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ICC/IF | (1) |
Use a concentration of 5 µg/ml.
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ChIP | (3) |
Use 2-5 µg for 25 µg of chromatin.
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ELISA |
Use at an assay dependent concentration.
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Flow Cyt (Intra) |
Use 1µg for 106 cells.
ab170190 - Mouse monoclonal IgG1, is suitable for use as an isotype control with this antibody. |
Notes |
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WB
Use a concentration of 1 - 5 µg/ml. Detects a band of approximately 17 kDa (predicted molecular weight: 15 kDa).Can be blocked with Human Histone H3 (tri methyl K4) peptide (ab1342). NOT SUITABLE for blocking with milk. Block in 5% BSA for 1 hour. Our labs have investigated the blocking conditions for this antibody and found that milk significantly decreases the signal and is therefore not a suitable blocking agent for this antibody (see Western Blot image). |
ICC/IF
Use a concentration of 5 µg/ml. |
ChIP
Use 2-5 µg for 25 µg of chromatin. |
ELISA
Use at an assay dependent concentration. |
Flow Cyt (Intra)
Use 1µg for 106 cells. ab170190 - Mouse monoclonal IgG1, is suitable for use as an isotype control with this antibody. |
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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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 25 µg of chromatin, 2 µg of ab12209 (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). Primers and probes are located in the first kb of the transcribed region.
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Ab12209 staining Histone H3 (Tri Methyl K4) in Mouse hair follicle DSCs by ICC/IF (Immunocytochemistry/Immunofluorescence). Cells were fixed with 3.7% paraformaldehyde, permeabilised with 0.2% Triton X-100 and blocked with 10% normal goat serum in PBS. Samples were incubated with primary antibody at 1:200 dilution. An Alexa Fluor ® 568 conjugated goat anti-mouse IgG was used as a secondary antibody.
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All lanes : Anti-Histone H3 (tri methyl K4) antibody [mAbcam12209] - ChIP Grade (ab12209) at 2 µg/ml
Lane 1 : Calf Thymus Histone Preparation Nuclear Lysate
Lane 2 : Calf Thymus Histone Preparation Nuclear Lysate withHuman Histone H3 (unmodified) peptide (ab7228) at 0.25 µg/ml
Lane 3 : Calf Thymus Histone Preparation Nuclear Lysate with Human Histone H3 (mono methyl K4) peptide (ab1340) at 0.25 µg/ml
Lane 4 : Calf Thymus Histone Preparation Nuclear Lysate with Human Histone H3 (di methyl K4) peptide (ab7768) at 0.25 µg/ml
Lane 5 : Calf Thymus Histone Preparation Nuclear Lysate withHuman Histone H3 (tri methyl K4) peptide (ab1342) at 0.25 µg/ml
Lane 6 : Calf Thymus Histone Preparation Nuclear Lysate with Human Histone H3 (mono methyl K9) peptide (ab1771) at 0.25 µg/ml
Lane 7 : Calf Thymus Histone Preparation Nuclear Lysate withHuman Histone H3 (di methyl K9) peptide (ab1772) at 0.25 µg/ml
Lane 8 : Calf Thymus Histone Preparation Nuclear Lysate withHuman Histone H3 (tri methyl K9) peptide (ab1773) at 0.25 µg/ml
Lane 9 : Calf Thymus Histone Preparation Nuclear Lysate with Human Histone H3 (mono methyl K27) peptide (ab1780) at 0.25 µg/ml
Lane 10 : Calf Thymus Histone Preparation Nuclear Lysate withHuman Histone H3 (di methyl K27) peptide (ab1781) at 0.25 µg/ml
Lane 11 : Calf Thymus Histone Preparation Nuclear Lysate withHuman Histone H3 (tri methyl K27) peptide (ab1782) at 0.25 µg/ml
Lysates/proteins at 0.5 µg per lane.
Secondary
All lanes : Goat polyclonal to Mouse IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution
Performed under reducing conditions.
Predicted band size: 15 kDa
Observed band size: 17 kDa why is the actual band size different from the predicted?
Exposure time: 16 minutes -
ICC/IF image of ab12209 stained human HeLa cells. The cells were 4% PFA fixed (10 min), permabilised in 0.1% PBS-Tween (20 min) and incubated with the antibody (ab12209, 5µg/ml) for 1h at room temperature. 1%BSA / 10% normal goat serum / 0.3M glycine was used to block non-specific protein-protein interactions. The secondary antibody (green) was Alexa Fluor® 488 goat anti-mouse IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red). DAPI was used to stain the cell nuclei (blue).
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ELISA using ab12209 at varying antibody concentrations.
Curve_SPL4 indicates binding to the tri methyl K4 peptide ab1342. In addition, SPL3 indicates partial binding to the di methyl K4 peptide ab7768. There is very weak cross-reactivity with the mono methyl K4 peptide ab1340 (Curve_SPL2).
Binding to the following peptides was not seen:
SPL1 unmodified Histone H3, SPL5 Histone H3 mono methyl K9, SPL6 Histone H3 di methyl K9, SPL7 Histone H3 tri methyl K9, SPL8 Histone H3 mono methyl K27, SPL9 Histone H3 di methyl K27, SPL10 Histone H3 tri methyl K27. -
Lane 1 : Anti-Histone H3 (tri methyl K4) antibody [mAbcam12209] - ChIP Grade (ab12209) at 1 µg/ml (BLOCKED WITH 5% BSA)
Lane 2 : Anti-Histone H3 (tri methyl K4) antibody [mAbcam12209] - ChIP Grade (ab12209) at 1 µg/ml (BLOCKED WITH 5% MILK)
All lanes : Calf Thymus Histone Preparation Nuclear Lysate
Lysates/proteins at 0.5 µg per lane.
Secondary
All lanes : Goat polyclonal to Mouse IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution
Performed under reducing conditions.
Predicted band size: 15 kDa
Observed band size: 17 kDa why is the actual band size different from the predicted?
Exposure time: 12 minutes -
Overlay histogram showing HeLa cells stained with ab12209 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab12209, 1µg/1x106 cells) for 30 min at 22ºC. The secondary antibody used was a goat anti-mouse DyLight® 488 (IgG; H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed.
Datasheets and documents
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SDS download
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Datasheet download
References (44)
ab12209 has been referenced in 44 publications.
- Xiao Q et al. Regulation of KDM5C stability and enhancer reprogramming in breast cancer. Cell Death Dis 13:843 (2022). PubMed: 36192394
- Davis JL et al. Single-cell multiomics reveals the complexity of TGFβ signalling to chromatin in iPSC-derived kidney organoids. Commun Biol 5:1301 (2022). PubMed: 36435939
- Ma YS et al. KDM5A silencing transcriptionally suppresses the FXYD3-PI3K/AKT axis to inhibit angiogenesis in hepatocellular cancer via miR-433 up-regulation. J Cell Mol Med 25:4040-4052 (2021). PubMed: 33621431
- Layden HM et al. A protocol for rapid degradation of endogenous transcription factors in mammalian cells and identification of direct regulatory targets. STAR Protoc 2:100530 (2021). PubMed: 34041503
- Guo JC et al. KDM5B promotes self-renewal of hepatocellular carcinoma cells through the microRNA-448-mediated YTHDF3/ITGA6 axis. J Cell Mol Med 25:5949-62 (2021). PubMed: 33829656