Anti-Histone H3 (methylated) antibody [6F12-H4] (ab71604)
- Product nameAnti-Histone H3 (methylated) antibody [6F12-H4]See all Histone H3 (methylated) primary antibodies ...
- DescriptionMouse monoclonal [6F12-H4] to Histone H3 (methylated)
- SpecificityIn Western blot ab71604 is blocked by tri methyl K9 and di methyl K9 peptides. It is not blocked by non-modified peptides or by methylated K4 or K27 peptides. Previous batches of ab71604 have generated a ChIP profile consistant with a Histone H3(tri methyl K9) antibody whereas our latest batch displays a profile more consistant with a Histone H3(di methyl K9) antibody. Please contact Scientific Support for further information.
- Tested applicationsFlow Cyt, WB, ICC/IF, IHC-P, ELISA more details
- Species reactivityReacts with: Cow, Human
Predicted to work with: Mouse, Rat, Rabbit, Hamster, Pig, Saccharomyces cerevisiae, Fruit fly (Drosophila melanogaster), Non Human Primates, a wide range of other species
Synthetic peptide of Human Histone H3, tri methylated at K9.
- Positive controlThis antibody gave a positive signal in a calf thymus histone preparation. This antibody gave a positive result in IHC in the following FFPE tissue: Human normal skin.
- Storage instructionsShipped at 4°C. Upon delivery aliquot and store at -20°C or -80°C. Avoid repeated freeze / thaw cycles.
- Storage bufferPreservative: 0.02% Sodium Azide
Constituents: PBS, pH 7.4
- Concentration information loading...
- PurityIgG fraction
- Clonality Monoclonal
- Clone number6F12-H4
- Research Areas
Our Abpromise guarantee covers the use of ab71604 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|Flow Cyt||Flow Cyt: Use 1µg for 106 cells.|
|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 - tri methyl K9 (ab1773).|
|ICC/IF||ICC/IF: Use a concentration of 1 - 5 µg/ml.|
|IHC-P||IHC-P: Use a concentration of 1 µg/ml.|
|ELISA||ELISA: Use at an assay dependent dilution.|
- 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: 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: 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
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Anti-Histone H3 (methylated) antibody [6F12-H4] images
All lanes : Anti-Histone H3 (methylated) antibody [6F12-H4] (ab71604) at 1 µg/ml
Lane 1 : Calf Thymus Histone Preparation Nuclear Lysate (ab121)
Lane 2 : Calf Thymus Histone Preparation Nuclear Lysate (ab121) with
Histone H3 peptide - unmodified (ab7228) at 2 µg/ml
Lane 3 : Calf Thymus Histone Preparation Nuclear Lysate (ab121) with
Histone H3 peptide - mono methyl K4 (ab1340) at 2 µg/ml
Lane 4 : Calf Thymus Histone Preparation Nuclear Lysate (ab121) with
Histone H3 peptide - di methyl K4 (ab7768) at 2 µg/ml
Lane 5 : Calf Thymus Histone Preparation Nuclear Lysate (ab121) with
Histone H3 peptide - tri methyl K4 (ab1342) at 2 µg/ml
Lane 6 : Calf Thymus Histone Preparation Nuclear Lysate (ab121) with
Histone H3 peptide - mono methyl K9 (ab1771) at 2 µg/ml
Lane 7 : Calf Thymus Histone Preparation Nuclear Lysate (ab121) with
Histone H3 peptide - di methyl K9 (ab1772) at 2 µg/ml
Lane 8 : Calf Thymus Histone Preparation Nuclear Lysate (ab121) with
Histone H3 peptide - tri methyl K9 (ab1773) at 2 µg/ml
Lane 9 : Calf Thymus Histone Preparation Nuclear Lysate (ab121) with
Histone H3 peptide - mono methyl K27 (ab1780) at 2 µg/ml
Lane 10 : Calf Thymus Histone Preparation Nuclear Lysate (ab121) with
Histone H3 peptide - di methyl K27 (ab1781) at 2 µg/ml
Lane 11 : Calf Thymus Histone Preparation Nuclear Lysate (ab121) with
Histone H3 peptide - tri methyl K27 (ab1782) at 2 µg/ml
Lysates/proteins at 0.5 µg per lane.
Goat polyclonal Secondary Antibody to Mouse IgG - H&L (HRP), pre-adsorbed (ab97040) at 1/10000 dilution
developed using the ECL technique
Performed under reducing conditions.
Predicted band size : 15 kDa
Exposure time : 3 minutes
ICC/IF image of ab71604 stained HeLa cells. The cells were 4% PFA fixed (10 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 (ab71604, 1µg/ml) overnight at +4°C. 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) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue). This antibody also gave a positive result in 4% PFA fixed (10 min) Hek293, MCF7 and HepG2 cells at 5µg/ml, and in 100% Methanol fixed (5 min) HeLa, Hek293, HepG2 and MCF7 cells at 5µg/ml.
IHC image of Histone H3 (tri methyl K9, di methyl K9) staining in Human normal skin formalin fixed paraffin embedded tissue section, performed on a Leica 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 ab71604, 1µg/ml, for 15 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.
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.
Overlay histogram showing HeLa cells stained with ab71604 (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 (ab71604, 1µg/1x106 cells) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse 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.
All batches of ab71604 are tested in Peptide Array against peptides to different Histone H3 modifications at 2µg/ml. 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 K9 peptide (ab1773) and di methyl K9 peptide (ab1772), indicating that this antibody recognises both modifications. Partial cross reactivity is observed with mono methyl K9 peptide (ab1771)
- ab1340 - Histone H3 - mono methyl K4
- ab7768 - Histone H3 - di 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
References for Anti-Histone H3 (methylated) antibody [6F12-H4] (ab71604)
ab71604 has not yet been referenced specifically in any publications.