Anti-Histone H3 (methylated) antibody - ChIP Grade (ab3594)
- Product nameAnti-Histone H3 (methylated) antibody - ChIP GradeSee all Histone H3 (methylated) primary antibodies ...
- DescriptionRabbit polyclonal to Histone H3 (methylated) - ChIP Grade
- SpecificitySpecific to Histone H3 K79 Di-methyl. In blocking experiments ab3594 is partially blocked by mono- or tri-methyl peptides.
- Tested applicationsChIP, ChIP/Chip, ICC/IF, WB, CHIPseq, IHC-P more details
- Species reactivityReacts with: Mouse, Cow, Human, Saccharomyces cerevisiae, Caenorhabditis elegans
Predicted to work with: a wide range of other species, all Mammals, Common Wheat (Triticum aestivum)
Synthetic peptide conjugated to KLH derived from within residues 50 to the C-terminus of Human Histone H3, di methylated at K79.
(Peptide available as ab4556.)
- Positive controlCalf Thymus Histone Preparation, Hela whole cell extract
- 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
Note: 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
- Clonality Polyclonal
- Research Areas
Our Abpromise guarantee covers the use of ab3594 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.|
|ChIP/Chip||ChIP/Chip: Use at an assay dependent dilution.|
|ICC/IF||ICC/IF: Use a concentration of 1 µg/ml.|
|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 - di methyl K79 (ab4556).|
|CHIPseq||CHIPseq: Use at an assay dependent concentration. PubMed: 22196736|
|IHC-P||IHC-P: Use a concentration of 1 µg/ml. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.|
- 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.
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.
- 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
- Omim: 601128 Human
- SwissProt: Q10453 Caenorhabditis elegans
- SwissProt: P08898 Caenorhabditis elegans
- SwissProt: P68432 Cow
- SwissProt: Q93081 Human
- SwissProt: Q16695 Human
- SwissProt: P68431 Human
- SwissProt: P68433 Mouse
- 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
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Anti-Histone H3 (methylated) antibody - ChIP Grade images
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 ab3594 (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.
IHC image of Histone H3 (di methyl K79) staining in human breast carcinoma FFPE section, performed on a BondTM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab3594, 1µg/ml, for 8 mins 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.
All lanes : Anti-Histone H3 (methylated) antibody - ChIP Grade (ab3594) at 1 µg/ml
Lane 1 : Calf thymus histone lysate
Lane 2 : Calf thymus histone lysate with
Histone H3 peptide - mono methyl K79 (ab4555) at 1 µg/ml
Lane 3 : Calf thymus histone lysate with
Histone H3 peptide - di methyl K79 (ab4556) at 1 µg/ml
Lane 4 : Calf thymus histone lysate with
Histone H3 peptide - tri methyl K79 (ab4557) at 1 µg/ml
Lane 5 : Calf thymus histone lysate with
Histone H4 peptide - di methyl K79 (ab4560) at 1 µg/ml
Lane 6 : Calf thymus histone lysate with
Histone H3 peptide - di methyl K9 (ab1772) at 1 µg/ml
Lane 7 : Calf thymus histone lysate with
Histone H3 peptide - unmodified (ab4558) at 1 µg/ml
Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (HRP) (ab6721) at 1/5000 dilution
Predicted band size : 15 kDa
Lane 3 shows that there is complete blocking with the immunsing peptide (ab4556). Lanes 2 and 4 show that there is slight cross reactivity with the Histone H3 mono methyl K79 and tri methyl K79 modification peptides.
ICC/IF image of ab3594 stained HeLa cells. The cells were 100% methanol fixed (5 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab3594, 1µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM. This antibody also gave a positive result in 100% methanol fixed (5 min) Hek293, HepG2 and MCF7 cells at 1µg/ml, and in 4% PFA fixed (10 min) HeLa, Hek293, HepG2 and MCF7 cells at 1µg/ml.
This image was kindly supplied as part of the review submitted by Dr Geza Fejes-Toth.
References for Anti-Histone H3 (methylated) antibody - ChIP Grade (ab3594)
This product has been referenced in:
- Wu H et al. Aqp5 is a new transcriptional target of Dot1a and a regulator of Aqp2. PLoS One 8:e53342 (2013). ICC/IF ; Mouse . Read more (PubMed: 23326416) »
- Youn MY et al. JMJD5, a Jumonji C (JmjC) Domain-containing Protein, Negatively Regulates Osteoclastogenesis by Facilitating NFATc1 Protein Degradation. J Biol Chem 287:12994-3004 (2012). WB ; Human . Read more (PubMed: 22375008) »