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Products:Epigenetics and Nuclear Signaling >> Histones >> H3 >> Methylated
Overview
Product name
Anti-Histone H3 (tri methyl K4) antibody - ChIP Grade
See all Histone H3 products (41) ...
Description
Rabbit polyclonal to Histone H3 (tri methyl K4) - ChIP Grade
Specificity
ab8580 detects a 17 kDa band in Western Blot which is completely blocked by addition of Histone H3 tri methyl K4 peptide. Partial blocking is observed with addition of Histone H3 di methyl K4 peptide. We recommend determining optimal dilutions/concentrations for peptide blocking studies. Modification specificity is determined by Peptide Array. ab8580 binds strongly to Histone H3 tri methyl K4, and partially to Histone H3 di methyl K4 peptides. Minimal reactivity is observed with mono methyl K4, unmodified, mono, di or tri methyl K9 or mono, di or tri methyl K27 peptides (please see data below). This antibody reacts with tri methylated K4 within a sequence found in all mammals and a wide range of other species, including S. cerevisiae, S. pombe, N. crassa, Aspergillus nidulans, D. melanogaster, S. ocellaris, C. reinhardtii, C. elegans, Arabidopsis thaliana, Chicken, Xenopus, Zebrafish and Tobacco. The antibody will react with any species where the modification is present.
Tested applications
ChIP/Chip, PepArr, ICC, ChIP, WB, IHC-Fr, IP, CHIPseq, Flow Cyt, ICC/IF, IHC-Pmore details
Cross reactivity
Reacts with
Mouse, Rat, Rabbit, Human, Pig, Saccharomyces cerevisiae, Tetrahymena sp., Arabidopsis thaliana, Caenorhabditis elegans, Fruit fly (Drosophila melanogaster), Zebrafish, Trypanosoma cruzi
Predicted to work with
Cow, Indian Muntjac, Oikopleura - a pelagic tunicate, Plants, all Mammals
Immunogen
Synthetic peptide conjugated to KLH derived from within residues 1 - 100 of Human Histone H3, tri methylated at K4.
(Peptide available as ab1342.)
Positive control
Calf Thymus Histone Preparation
Properties
Form
Liquid
Storage instructions
Store at +4°C short term (1-2 weeks). Aliquot and store at -20°C or -80°C. Avoid repeated freeze / thaw cycles.
Storage buffer
pH: 7.40
Preservative: 0.02% Sodium azide
Constituents: 1% BSA, PBS
Concentration
Concentration information loading...
Purity
Immunogen affinity purified
Primary antibody 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. The 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.
Clonality
Polyclonal
Isotype
IgG
Research areas
Epigenetics and Nuclear Signaling >> ChIP'ing antibodies >> ChIP'ing antibodies
Epigenetics and Nuclear Signaling >> Histones >> H3 >> Methylated
Applications
Show applications key
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.
ChIP/Chip: Use at an assay dependent dilution.
PepArr: Use a concentration of 0.2 - 2 µg/ml.
ICC: Use at an assay dependent dilution.
ChIP: Use 2 µg for 25 µg of chromatin.
WB: Use a concentration of 1 µg/mlDetects a band of approximately 17 kDa (predicted molecular weight: 15 kDa).Can be blocked with Histone H3 peptide - tri methyl K4 (ab1342).
IHC-Fr
IHC-Fr: 1/500IHC-Fr: 1/500
IP: Use at an assay dependent dilution.
CHIPseq: Use at an assay dependent dilution. (PubMed: 20952408)
Flow Cyt: Use at an assay dependent dilution.
ICC/IF: 1/100 - 1/5000.
IHC-P: Use at an assay dependent dilution. (PubMed: 17634443)
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.
Target information above from: UniProt accessionP68431
The UniProt Consortium
The Universal Protein Resource (UniProt) in 2010
Nucleic Acids Res. 38:D142-D148 (2010).
Alternative names
- Histone H3.1 antibody
- FLJ92264 antibody
- H3 histone antibody
see all
Database links
- Entrez Gene: 176359 Caenorhabditis elegans
- Entrez Gene: 326601 Cow
- Entrez Gene: 8350 Human
- Entrez Gene: 8351 Human
- Entrez Gene: 8352 Human
- Entrez Gene: 8353 Human
- Entrez Gene: 8354 Human
- Entrez Gene: 8355 Human
see all
Anti-Histone H3 (tri methyl K4) antibody - ChIP Grade images:
ChIP - Histone H3 (tri methyl K4) antibody - ChIP Grade (ab8580)
Chromatin was prepared from U2OS cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10 min. 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.
Peptide Array - 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.
- 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
Western blot - 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 polyclonal to Rabbit 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 : 8 minutes
Western blot - Histone H3 (tri methyl K4) antibody - ChIP Grade (ab8580)
All lanes : Anti-Histone H3 (tri methyl K4) antibody - ChIP Grade (ab8580) at 1/5000 dilution
Lane 1 : S. cerevisiae whole cell lysate with WT
Lane 2 : S. cerevisiae whole cell lysate with set1
Lane 3 : S. cerevisiae whole cell lysate with bre2
Lane 4 : S. cerevisiae whole cell lysate with sdc1
Lane 5 : S. cerevisiae whole cell lysate with shg1
Lane 6 : S. cerevisiae whole cell lysate with spp1
Lane 7 : S. cerevisiae whole cell lysate with swd1
Lane 8 : S. cerevisiae whole cell lysate with swd3
Lysates/proteins at 40 µg per lane.
Performed under reducing conditions.
Predicted band size : 15 kDa
Observed band size : 15 kDa
Protein resolved on 15% SDS-PAGE gel. After transfer to PVDF membrane, blots were blocked in 1X PBS, 0.1% Tween-20, and 5% milk. ab1791 was diluted in 5 ml blocking buffer at 1/5000. Blots plus primary antibodies were either incubated overnight at 4C or at RT for 2 hr. Blots were washed 6X for 10 min each in PBS with 0.1% Tween-20 before addition of secondary antibodies. Secondary antibodies were diluted 1/2,000 in blocking buffer and incubated with blots for 2 hr at RT. Secondary blots were washed 4X for 10 min each in PBS with 0.1% Tween-20 and 2X for 10 min each in PBS.
John E. Mueller and J. Ruth German (Mary Bryk lab)
Immunocytochemistry - Histone H3 (tri methyl K4) antibody - ChIP Grade (ab8580)
ab8580 staining cultured human primary fibroblasts by ICC. Cells were PFA fixed and permeabilized in TritonX100 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. A FITC-conjugated rabbit anti-rabbit IgG antibody was used as the secondary.
This image is courtesy of an Abreview submitted by Dr Eva Bartova
Immunofluorescence - Histone H3 (tri methyl K4) antibody - ChIP Grade (ab8580)
Staining (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).
Kirk McManus in the lab of Michael Hendzel, University of Alberta
Immunofluorescence - Histone H3 (tri methyl K4) antibody - ChIP Grade (ab8580)
IF of primary cultures of bladder cancer which arise from urothelial cells to determine whether the alterations in chromatin during cancer development would be enhanced by the histone modification antibodies. Both cell types gave very weak staining with the trimethyl-K9-H3 antibody.
Wolfgang Schulz
Immunofluorescence - Histone H3 (tri methyl K4) antibody - ChIP Grade (ab8580)
Mouse zygotes stained with the Tri-Methyl K4 histone H3 antibody
(green) and DNA (blue). This modification is readily detected in the two
pronuclei of the zygote.
This image was kindly supplied as part of the review submitted by Dr Maria Elena Torres Padilla (University of Cambridge, UK).
Immunofluorescence - Histone H3 (tri methyl K4) antibody - ChIP Grade (ab8580)
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.
Ahmad Khalil and Daniel Driscoll, University of Florida College of Medicine.
Protocols
References for Anti-Histone H3 (tri methyl K4) antibody - ChIP Grade (ab8580)
This product has been referenced in:
- Sauer Set al. T cell receptor signaling controls Foxp3 expression via PI3K, Akt, and mTOR. Proc Natl Acad Sci U S A : (2008). ChIP; Fruit fly (Drosophila melanogaster).Read more (PubMed: 18509048) »
- Roh TYet al. The genomic landscape of histone modifications in human T cells. Proc Natl Acad Sci U S A 103:15782-7 (2006). ChIP; Human.Read more (PubMed: 17043231) »
See all 224 publications for this product
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Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"