Anti-Histone H3 (tri methyl K4) antibody (ab8580)
Overview
- Product nameAnti-Histone H3 (tri methyl K4) antibodySee all Histone H3 (methylated) primary antibodies ...
- DescriptionRabbit polyclonal to Histone H3 (tri methyl K4)
- Specificityab8580 detects a 17 kDa band in single lane Western Blot. Peptide inhibition in Western Blot hasn't been processed. 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 applicationsChIP/Chip, PepArr, ICC, ChIP, WB, IHC-Fr, IP, CHIPseq, Flow Cyt, ICC/IF, IHC-P more details
- Species reactivityReacts with: Mouse, Rat, Rabbit, Human, Pig, Saccharomyces cerevisiae, Tetrahymena sp., Arabidopsis thaliana, Caenorhabditis elegans, Fruit fly (Drosophila melanogaster), Zebrafish, Trypanosoma cruzi, Marmoset (common)
Predicted to work with: Cow, Indian Muntjac, Oikopleura - a pelagic tunicate, Plants, all Mammals
- Immunogen
Synthetic peptide corresponding to Human Histone H3 aa 1-100 (tri methyl K4) conjugated to Keyhole Limpet Haemocyanin (KLH).
(Peptide available asab92374) - Positive controlCalf Thymus Histone Preparation
- 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. 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.
Properties
- FormLiquid
- Storage instructionsStore at +4°C short term (1-2 weeks). Aliquot and store at -20°C or -80°C. Avoid repeated freeze / thaw cycles.
- Storage bufferpH: 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... - PurityImmunogen 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
- IsotypeIgG
- Research Areas
Applications
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.
| Application | Notes |
|---|---|
| ChIP/Chip | ChIP/Chip: Use at an assay dependent dilution. |
| PepArr | PepArr: Use a concentration of 0.2 - 2 µg/ml. |
| ICC | ICC: Use at an assay dependent dilution. |
| ChIP | ChIP: Use 2 µg for 25 µg of chromatin. |
| WB | WB: Use a concentration of 1 µg/ml. Detects a band of approximately 17 kDa (predicted molecular weight: 15 kDa).Can be blocked with Histone H3 peptide (1-10) - tri methyl K4 (ab92374). |
| IHC-Fr | IHC-Fr: 1/500. |
| IP | IP: Use at an assay dependent dilution. |
| CHIPseq | CHIPseq: Use at an assay dependent dilution. PubMed: 20952408 |
| Flow Cyt | Flow Cyt: Use at an assay dependent dilution. |
| ICC/IF | ICC/IF: 1/100 - 1/5000. |
| IHC-P | IHC-P: Use at an assay dependent dilution. PubMed: 17634443 |
Target
- FunctionCore 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 similaritiesBelongs to the histone H3 family.
- Developmental stageExpressed 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 localizationNucleus. Chromosome.
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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
- Entrez Gene: 8356 Human
- Entrez Gene: 8357 Human
- Entrez Gene: 8358 Human
- Entrez Gene: 8968 Human
- Entrez Gene: 319152 Mouse
- Entrez Gene: 319153 Mouse
- Entrez Gene: 360198 Mouse
- Entrez Gene: 97908 Mouse
- Entrez Gene: 100364501 Rat
- Entrez Gene: 100365669 Rat
- Entrez Gene: 291159 Rat
- Entrez Gene: 679994 Rat
- Entrez Gene: 680511 Rat
- Entrez Gene: 682330 Rat
- Omim: 601128 Human
- SwissProt: P59226 Arabidopsis thaliana
- SwissProt: Q10453 Caenorhabditis elegans
- SwissProt: P08898 Caenorhabditis elegans
- SwissProt: P68432 Cow
- SwissProt: P02299 Fruit fly (Drosophila melanogaster)
- SwissProt: Q93081 Human
- SwissProt: Q16695 Human
- SwissProt: P68431 Human
- SwissProt: P68433 Mouse
- SwissProt: Q6LED0 Rat
- Unigene: 132854 Human
- Unigene: 247813 Human
- Unigene: 247814 Human
- Unigene: 248176 Human
- Unigene: 443021 Human
- Unigene: 484990 Human
- Unigene: 532144 Human
- Unigene: 533292 Human
- Unigene: 546315 Human
- Unigene: 586261 Human
- Unigene: 591778 Human
- Unigene: 221301 Mouse
- Unigene: 261657 Mouse
- Unigene: 377874 Mouse
- Unigene: 390558 Mouse
- Unigene: 397328 Mouse
- Unigene: 138090 Rat
see all
Target information above from: UniProt accession
P68431
The UniProt Consortium
The Universal Protein Resource (UniProt) in 2010
Nucleic Acids Res. 38:D142-D148 (2010)
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Alternative names
- FLJ92264 antibodyH3 histone antibodyH3 histone antibody
- H3 histone family, member A antibodyH3/a antibodyH3/b antibodyH3/c antibodyH3/d antibodyh3/f antibodyH3/h antibodyH3/i antibodyH3/j antibodyH3/k antibodyH3/l antibodyH31_HUMAN antibodyH3F1K antibodyH3F3 antibodyH3F3 antibodyH3FA antibodyH3FB antibodyH3FC antibodyH3FD antibodyH3FF antibodyH3FH antibodyH3FI antibodyH3FJ antibodyH3FK antibodyH3FL antibodyHIST1H3A antibodyHIST1H3B antibodyHIST1H3C antibodyHIST1H3D antibodyHIST1H3E antibodyHIST1H3F antibodyHIST1H3G antibodyHIST1H3H antibodyHIST1H3I antibodyHIST1H3J antibodyHIST3H3 antibodyHIST3H3 antibodyHistone 1, H3a antibodyHistone cluster 1, H3a antibodyHistone cluster 1, H3b antibodyHistone cluster 1, H3c antibodyHistone cluster 1, H3d antibodyHistone cluster 1, H3e antibodyHistone cluster 1, H3f antibodyHistone cluster 1, H3g antibodyHistone cluster 1, H3i antibodyHistone cluster 1, H3j antibodyHistone H 3 antibodyHistone H3.1 antibodyHistone H3.1 antibodyHistone H3/a antibodyHistone H3/b antibodyHistone H3/c antibodyHistone H3/d antibodyHistone H3/f antibodyHistone H3/h antibodyHistone H3/i antibodyHistone H3/j antibodyHistone H3/k antibodyHistone H3/l antibody
see all
Anti-Histone H3 (tri methyl K4) antibody images
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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.
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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
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Western blot - Histone H3 (tri methyl K4) antibody - ChIP Grade (ab8580)Anti-Histone H3 (tri methyl K4) antibody (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 (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)
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Immunocytochemistry - 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 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.
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Immunofluorescence - Histone H3 (tri methyl K4) antibody - ChIP Grade (ab8580)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).
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Immunofluorescence - Histone H3 (tri methyl K4) antibody - ChIP Grade (ab8580)Wolfgang SchulzIF 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. -
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).
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Immunofluorescence - Histone H3 (tri methyl K4) antibody - ChIP Grade (ab8580)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. -
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H3 (tri methyl K4) antibody - ChIP Grade (ab8580)This image is courtesy of an abreview by Zachary Yu-Ching Lin.IHC-P image of Histone H3 (tri methyl K4) staining with ab8580 on tissue sections from adult marmoset testis. The sections were subjected to heat-mediated antigen retrieval using Dako antigen retrieval solution. The sections were blocked with 5% milk for 30 minutes at 25°C and then incubated with ab8580 (1/100 dilution) for 16 hours at 4°C. The secondary used was an Alexa-Fluor 555 conjugated goat anti-rabbit polyclonal.
Protocols
References for Anti-Histone H3 (tri methyl K4) antibody (ab8580)
This product has been referenced in:
- Wan M et al. The trithorax group protein Ash2l is essential for pluripotency and maintaining open chromatin in embryonic stem cells. J Biol Chem 288:5039-48 (2013). WB, ChIP ; Mouse . Read more (PubMed: 23239880) »
- Wu MF et al. SWI2/SNF2 chromatin remodeling ATPases overcome polycomb repression and control floral organ identity with the LEAFY and SEPALLATA3 transcription factors. Proc Natl Acad Sci U S A 109:3576-81 (2012). ChIP . Read more (PubMed: 22323601) »
