Anti-Histone H3 (tri methyl K4) antibody - ChIP Grade (ab8580)
Key features and details
- Rabbit polyclonal to Histone H3 (tri methyl K4) - ChIP Grade
- Suitable for: PepArr, ChIP, WB, IHC-P, ICC/IF
- Reacts with: Cow, Human
- Isotype: IgG
Overview
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Product name
Anti-Histone H3 (tri methyl K4) antibody - ChIP Grade
See all Histone H3 primary antibodies -
Description
Rabbit polyclonal to Histone H3 (tri methyl K4) - ChIP Grade -
Host species
Rabbit -
Tested Applications & Species
See all applications and species dataApplication Species ChIP HumanICC/IF HumanIHC-P HumanWB Cow
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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 asab92374) -
General notes
In immunofluorescence, a distinct property of tri methyl lysine 4 is its apparent 'ringing' of regions that appear as nucleoplasmic 'holes'. These represent the positions of splicing factor compartments, which often are easy to identify using only DNA stains in Indian muntjac fibroblasts. These splicing factor compartments are known to be preferentially associated with active genes and highly acetylated histone H3. This antibody, as expected, fails to stain heterochromatin (work by Kirk McManus, lab of Michael Hendzel).
The immunofluorescence results suggest this antibody is an exceptional euchromatin probe.
Learn about ChIP assay kits, other ChIP antibodies, protocols and more in the ChIP assay guide.
Abcam recommended secondaries - Goat Anti-Rabbit HRP (ab205718) and Goat Anti-Rabbit Alexa Fluor® 488 (ab150077). See other anti-rabbit secondary antibodies that can be used with this antibody.
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 the problem with our range of recombinant monoclonal antibodies and knockout edited cell lines for gold-standard validation.
One factor contributing to the crisis is the use of antibodies that are not suitable. This can lead to misleading results and the use of incorrect data informing project assumptions and direction. To help address this challenge, we have introduced an application and species grid on our primary antibody datasheets to make it easy to simplify identification of the right antibody for your needs.
Learn more here.
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
Constituent: PBS
Batches of this product that have a concentration < 1mg/ml may have BSA added 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... -
Purity
Immunogen affinity purified -
Clonality
Polyclonal -
Isotype
IgG -
Research areas
Associated products
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ChIP Related Products
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Compatible Secondaries
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Control 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
- Anti-Histone H3 antibody [mAbcam 10799] - ChIP Grade (ab10799)
- Histone H3 (K4) Methyltransferase Activity Quantification Assay Kit (ab113452)
- Histone Demethylase (H3K4) Activity Quantification Assay Kit (ab113455)
- Histone H3 (tri-methyl K4) Quantification Kit (Colorimetric) (ab115056)
- Prestained Protein Ladder - Broad molecular weight (10-245 kDa) (ab116028)
- Anti-Histone H3 (di methyl K9) antibody [mAbcam 1220] - ChIP Grade (ab1220)
- Human Histone H3 (tri methyl K9) peptide (ab1773)
- Human Histone H3 (tri methyl K27) peptide (ab1782)
- Anti-Histone H3 (tri methyl K27) antibody [mAbcam 6002] - ChIP Grade (ab6002)
- Anti-Histone H3 (di methyl K4) antibody - ChIP Grade (ab7766)
- Human Histone H3 (di methyl K4) peptide (ab7768)
- Anti-Histone H3 (mono methyl K4) antibody - ChIP Grade (ab8895)
Applications
The Abpromise guarantee
Our Abpromise guarantee covers the use of ab8580 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Tested applications are guaranteed to work and covered by our Abpromise guarantee.
Predicted to work for this combination of applications and species but not guaranteed.
Does not work for this combination of applications and species.
| Application | Species |
|---|---|
| ChIP |
Human
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| ICC/IF |
Human
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| IHC-P |
Human
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| WB |
Cow
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| All applications |
Mouse
Rat
Rabbit
Pig
Saccharomyces cerevisiae
Tetrahymena
Xenopus laevis
Arabidopsis thaliana
Caenorhabditis elegans
Drosophila melanogaster
Indian muntjac
Oikopleura
Plants
Zebrafish
Mammals
Trypanosoma cruzi
Common marmoset
Rice
Xenopus tropicalis
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| Application | Abreviews | Notes |
|---|---|---|
| PepArr |
Use a concentration of 0.2 - 0.02 µg/ml.
Slight cross reactivity is observed with the Histone H3 - di methyl K4 modification. Optimisation is recommended to avoid array signal saturation. |
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| ChIP | (26) |
Use 2 µg for 25 µg of chromatin.
We recommend GAPDH positive control ChIP primer pair ab267832 as positive control. |
| WB | (19) |
Use a concentration of 1 µg/ml. Detects a band of approximately 17 kDa (predicted molecular weight: 15 kDa).
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| IHC-P | (8) |
Use at an assay dependent concentration.
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| ICC/IF | (19) |
Use a concentration of 1 µg/ml.
1/100 - 1/5000 |
| Notes |
|---|
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PepArr
Use a concentration of 0.2 - 0.02 µg/ml. Slight cross reactivity is observed with the Histone H3 - di methyl K4 modification. Optimisation is recommended to avoid array signal saturation. |
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ChIP
Use 2 µg for 25 µg of chromatin. We recommend GAPDH positive control ChIP primer pair ab267832 as positive control. |
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WB
Use a concentration of 1 µg/ml. Detects a band of approximately 17 kDa (predicted molecular weight: 15 kDa). |
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IHC-P
Use at an assay dependent concentration. |
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ICC/IF
Use a concentration of 1 µg/ml. 1/100 - 1/5000 |
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 (tri methyl K4) antibody - ChIP Grade (ab8580)
Chromatin was prepared from U-2 OS (Human bone osteosarcoma epithelial cell line) cells according to the Abcam X-ChIP protocol.
Cells were fixed with formaldehyde for 10 minutes. The ChIP was performed with 25 µg of chromatin, 2 µg of ab8580 (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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Western blot - Anti-Histone H3 (tri methyl K4) antibody - ChIP Grade (ab8580)Anti-Histone H3 (tri methyl K4) antibody - ChIP Grade (ab8580) at 1 µg/ml + Calf thymus histone preparation (nuclear lysate) at 0.5 µg
Secondary
Goat Anti-Rabbit IgG (H+L) HRP- conjugated antibody at 1/50000 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: 8 minutes -
Immunocytochemistry/ Immunofluorescence - Anti-Histone H3 (tri methyl K4) antibody - ChIP Grade (ab8580)This image is courtesy of Ahmad Khalil and Daniel Driscoll, University of Florida College of Medicine.Human female lymphoblast immunostained with ab8580 (1:100) (yellowish green) specific for histone H3 lysine 4 (H3-K4) trimethylation; the DNA is stained red with propidium iodide (PI).
Note the inactive X chromosome (arrow) and pericentromeric heterochromatin are largely devoid of this modification.
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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H3 (tri methyl K4) antibody - ChIP Grade (ab8580)IHC image of ab8580 staining Histone H3 (tri methyl K4) in human colon formalin-fixed paraffin-embedded tissue sections*, performed on a Leica Bond.
The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6, epitope retrieval solution 1) for 20 minutes. The section was then incubated with ab8580, 1/500 dilution, for 15 minutes at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.
No primary antibody was used in the negative control (inset).
For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.
*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre
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Immunocytochemistry/ Immunofluorescence - Anti-Histone H3 (tri methyl K4) antibody - ChIP Grade (ab8580)This image is courtesy of an Abreview submitted by Dr Eva Bartovaab8580 staining cultured human primary fibroblasts by ICC.
Cells were fixed in PFA and permeabilized in Triton X-100 and saponin prior to blocking with 1% BSA for 1 hour at RT. The primary antibody was diluted 1/100 and incubated with the sample for 16 hours at 4°C. An FITC-conjugated rabbit anti-rabbit IgG antibody was used as the secondary.
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Immunocytochemistry/ Immunofluorescence - Anti-Histone H3 (tri methyl K4) antibody - ChIP Grade (ab8580)ab8580 staining Histone H3 (tri methyl K4) in HeLa (Human epithelial cell line from cervix adenocarcinoma) cells.
All cells were fixed with 100% methanol (5 minutes) and then blocked in 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1 hour. The cells were then incubated with ab4729 at 1/1000 and ab7291 at 1µg/ml overnight at +4°C, followed by a further incubation at room temperature for 1 hour with goat anti-rabbit Alexa-Fluor®488 secondary (ab150077) at 2 μg/ml (shown in green) and goat anti-mouse Alexa-Fluor®594 secondary (ab150120) at 2 μg/ml (shown in pseudo color red). Nuclear DNA was labeled in blue with DAPI.
Negative controls: 1– Rabbit primary and anti-mouse secondary antibody; 2 – Mouse primary antibody and anti-rabbit secondary antibody. Controls 1 and 2 indicate that there is no non-specific reaction between primary and secondary antibodies used.
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Peptide Array - Anti-Histone H3 (tri methyl K4) antibody - ChIP Grade (ab8580)All batches of ab8580 are tested in Peptide Array against peptides to different Histone H3 modifications. Six dilutions of each peptide are printed on to the Peptide Array in triplicate and results are averaged before being plotted on to a graph. Results show strong binding to Histone H3 - tri methyl K4 peptide (ab1342), indicating that this antibody specifically recognises the Histone H3 - tri methyl K4 modification. Slight cross reactivity is observed with the Histone H3 - di methyl K4 modification. Optimization is recommended to avoid array signal saturation.
- ab1340 - Histone H3 - mono methyl K4
- ab1342 - Histone H3 - tri methyl K4
- ab1771 - Histone H3 - mono methyl K9
- ab1772 - Histone H3 - di methyl K9
- ab1773 - Histone H3 - tri methyl K9
- ab1780 - Histone H3 - mono methyl K27
- ab1781 - Histone H3 - di methyl K27
- ab1782 - Histone H3 - tri methyl K27
- ab7228 - Histone H3 - unmodified
- ab7768 - Histone H3 - di methyl K4
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ChIP - Anti-Histone H3 (tri methyl K4) antibody - ChIP Grade (ab8580)This image is courtesy of an anonymous AbreviewChromatin was prepared from human cell lysate - nuclear B cells according to the Abcam X-ChIP protocol.
Cells were fixed with formaldehyde for 10 minutes. The ChIP was performed with 0.5 µg of ab8580 per µg chromatin in ChIP Buffer fot 16 hours at 4°C. The immunoprecipitated DNA was quantified by real time PCR (Taqman approach). Primers and probes are located in the first kb of the transcribed region. Negative control: IgG and Gene Desert. Positive control: GAPDH Promoter.
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Immunocytochemistry/ Immunofluorescence - Anti-Histone H3 (tri methyl K4) antibody - ChIP Grade (ab8580)This image is courtesy of Kirk McManus in the lab of Michael Hendzel, University of AlbertaStaining (green) with the anti-trimethyl Lysine K4 of Histone H3 antibody (ab8580) shows ringing of regions that appear as nucleoplasmic holes. These represent the positions of splicing factor compartments, which are preferentially associated with active genes and highly acetylated histone H3.
The antibody, as expected, fails to stain heterochromatin (red).
Datasheets and documents
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SDS download
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Datasheet download
References (1513)
ab8580 has been referenced in 1513 publications.
- Han M et al. Therapeutic implications of altered cholesterol homeostasis mediated by loss of CYP46A1 in human glioblastoma. EMBO Mol Med 12:e10924 (2020). PubMed: 31777202
- Li Q et al. p53 Integrates Temporal WDR5 Inputs during Neuroectoderm and Mesoderm Differentiation of Mouse Embryonic Stem Cells. Cell Rep 30:465-480.e6 (2020). PubMed: 31940490
- Domingues AF et al. Loss of Kat2a enhances transcriptional noise and depletes acute myeloid leukemia stem-like cells. Elife 9:N/A (2020). PubMed: 31985402
- Castelijns B et al. Hominin-specific regulatory elements selectively emerged in oligodendrocytes and are disrupted in autism patients. Nat Commun 11:301 (2020). PubMed: 31949148
- Karagianni P et al. Bookmarking by Non-pioneer Transcription Factors during Liver Development Establishes Competence for Future Gene Activation. Cell Rep 30:1319-1328.e6 (2020). PubMed: 32023452