![Western blot - Histone H3 (di+tri methyl K4) antibody [mAbcam 6000] (ab6000) image](#ab6000_1.jpg)
![Immunocytochemistry/ Immunofluorescence - Histone H3 (di+tri methyl K4) antibody [mAbcam 6000] - ChIP Grade (ab6000) image](#ab6000_2.jpg)
![ChIP - Histone H3 (di+tri methyl K4) antibody [mAbcam 6000] - ChIP Grade (ab6000) image](#ab6000_8.jpg)
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Products:Epigenetics and Nuclear Signaling >> Histones >> H3 >> Methylated
Anti-Histone H3 (di+tri methyl K4) antibody [mAbcam 6000] - ChIP Grade (ab6000)
Overview
Product name
Anti-Histone H3 (di+tri methyl K4) antibody [mAbcam 6000] - ChIP Grade
See all Histone H3 products (41) ...
Description
Mouse monoclonal [mAbcam 6000] to Histone H3 (di+tri methyl K4) - ChIP Grade
Specificity
This antibody detects a band of the appropriate size when used in Western blotting on a calf thymus histone preparation (see image). It is specifically blocked by peptides corresponding to di methylated K4 of Histone H3 and to tri methylated K4. It is not blocked by peptides corresponding to mono methylated K4, mono methyl K27, di methyl K9, tri methyl K9 and unmodified K4. Thus, we believe that this antibody is specific for di and tri methylated K4 of Histone H3.
Tested applications
ICC/IF, IHC-P, WB, ICC, ChIPmore details
Cross reactivity
Reacts with
Mouse, Rabbit, Cow, Human, Fruit fly (Drosophila melanogaster), Rice
Predicted to work with
Rat, Chicken, Saccharomyces cerevisiae, Xenopus laevis, Arabidopsis thaliana, Caenorhabditis elegans, Schizosaccharomyces pombe, Neurospora crassa
Immunogen
Synthetic peptide derived from residues 1 - 100 of Human Histone H3, tri methylated at K4.
. Hybridomas were prepared and the resulting clones were positively screened by ELISA against the immunising peptide and negatively screened against the non-modified equivalent peptide .
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
Preservative: 0.02% Sodium Azide
Constituents: PBS, pH 7.4
Concentration
Concentration information loading...
Purity
IgG fraction
Clonality
Monoclonal
Clone number
mAbcam 6000
Myeloma
Sp2/0-Ag14
Isotype
IgG2b
Light chain type
kappa
Research areas
Epigenetics and Nuclear Signaling >> ChIP'ing antibodies >> ChIP'ing antibodies
Epigenetics and Nuclear Signaling >> Histones >> H3 >> Methylated
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Western blot - Histone H3 (di+tri methyl K4) antibody [mAbcam 6000] (ab6000)
![Western blot - Histone H3 (di+tri methyl K4) antibody [mAbcam 6000] (ab6000) image](/ps/datasheet/Images/6/ab6000/ab6000_1.jpg)
(enlarge)
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Immunocytochemistry/ Immunofluorescence - Histone H3 (di+tri methyl K4) antibody [mAbcam 6000] - ChIP Grade (ab6000)
![Immunocytochemistry/ Immunofluorescence - Histone H3 (di+tri methyl K4) antibody [mAbcam 6000] - ChIP Grade (ab6000) image](/ps/datasheet/Images/6/ab6000/ab6000_2.jpg)
(enlarge)
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ChIP - Histone H3 (di+tri methyl K4) antibody [mAbcam 6000] - ChIP Grade (ab6000)
![ChIP - Histone H3 (di+tri methyl K4) antibody [mAbcam 6000] - ChIP Grade (ab6000) image](/ps/datasheet/Images/6/ab6000/ab6000_8.jpg)
(enlarge)
Applications
Show applications key
Our Abpromise guarantee covers the use of ab6000 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
ICC/IF: Use a concentration of 1 µg/ml
IHC-P: Use at an assay dependent dilution. (PubMed: 20823885)
WB: 1/500Predicted molecular weight: 15.2 kDa.Can be blocked with Histone H3 peptide - tri methyl K4 (ab1342) or Histone H3 peptide - di methyl K4 (ab7768).
ICC
ICC: 1/200ICC: 1/200
ChIP: Use at an assay dependent dilution.
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: 427887 Chicken
- Entrez Gene: 326601 Cow
- Entrez Gene: 8350 Human
- Entrez Gene: 8351 Human
- Entrez Gene: 8352 Human
- Entrez Gene: 8353 Human
- Entrez Gene: 8354 Human
see all
Anti-Histone H3 (di+tri methyl K4) antibody [mAbcam 6000] - ChIP Grade images:
Western blot - Histone H3 (di+tri methyl K4) antibody [mAbcam 6000] (ab6000)
All lanes : Anti-Histone H3 (di+tri methyl K4) antibody [mAbcam 6000] - ChIP Grade (ab6000) at 1/500 dilution
Lane 1 : Histone Prep
Lane 2 : Histone Prep with Histone H3 peptide - mono methyl K4 (ab1340) at 1 µg/ml
Lane 3 : Histone Prep with Histone H3 peptide - di methyl K4 (ab7768) at 1 µg/ml
Lane 4 : Histone Prep with Histone H3 peptide - tri methyl K4 (ab1342) at 1 µg/ml
Lane 5 : Histone Prep with Histone H3 peptide - mono methyl K27 (ab1780) at 1 µg/ml
Lane 6 : Histone Prep with Histone H3 peptide - di methyl K9 (ab1772) at 1 µg/ml
Lane 7 : Histone Prep with Histone H3 peptide - tri methyl K9 (ab1773) at 1 µg/ml
Lane 8 : Histone Prep with Histone H3 peptide - unmodified (ab2903) at 1 µg/ml
Lysates/proteins at 1 µg per lane.
Secondary
Rabbit polyclonal Secondary Antibody to Mouse IgG - H&L (HRP) (ab6728) at 1/5000 dilution
Performed under reducing conditions.
Predicted band size : 15.2 kDa
Exposure time : 2 minutes
Immunocytochemistry/ Immunofluorescence - Histone H3 (di+tri methyl K4) antibody [mAbcam 6000] - ChIP Grade (ab6000)
Immunofluorescent imaging of human cells (U2OS) with ab6000 reveals a diffuse background nuclear staining corresponding to global dimethylation of K4, with multiple foci of brighter staining. The complete lack of nucleolar or cytoplasmic staining background confirms the high specificity of this antibody in this application.
IF was performed with a standard paraformaldehyde technique (fixed in PBS buffered PFH 4% for 5 minutes, permeabilised with 0.5% triton-PBS for 5 minutes, blocked with 5% milk / 0.2% tween for one hour. Primary antibody used at 1/200 in 5% milk / 0.2% TWEEN for one hour, secondary antibody Alexa 488 for 30 minutes. All blocking and incubation steps carried out at 37 degrees.
Due to the size constraints for images on our website, unfortunately we are unable to show a higher quality image.
Luke Hughes-Davies and Rhiannon Jade, Gurdon Institute, Cambridge, UK
ChIP - Histone H3 (di+tri methyl K4) antibody [mAbcam 6000] - ChIP Grade (ab6000)
Chromatin was prepared from U2OS cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10min. The ChIP was performed with 25 µg of chromatin, 2 µg of ab6000 (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.
Immunocytochemistry/ Immunofluorescence - Histone H3 (di+tri methyl K4) antibody [mAbcam 6000] - ChIP Grade (ab6000)
![Immunocytochemistry/ Immunofluorescence - Histone H3 (di+tri methyl K4) antibody [mAbcam 6000] - ChIP Grade (ab6000)](/ps/datasheet/images/6/ab6000/Histone-H3-Primary-antibodies-ab6000-2.jpg)
ICC/IF image of ab6000 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 (ab6000, 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) at a concentration of 1.43µM. This antibody also gave a positive result in 4% PFA fixed (10 min) HepG2 and MCF7 cells at 1µg/ml.
Immunofluorescence - Anti-Histone H3 (di+tri methyl K4) antibody [mAbcam 6000] - ChIP Grade (ab6000)
![Immunofluorescence - Anti-Histone H3 (di+tri methyl K4) antibody [mAbcam 6000] - ChIP Grade (ab6000)](/ps/datasheet/Images/6/ab6000/ab6000_3.jpg)
Left image shows human Hep cell monolayer fixed with 4% formaldehyde, permeabilized using 0.5% Triton X-100, blocked with 5% FCS in PBS (20 min) and incubated with ab6000 for 1 hr (diluted 1/200 in PBS plus 1% FCS). Detection with anti-mouse rhodamin labeled secondary antibody. Unstained areas represent nucleoli and heterochromatin as judged from DAPI overlay of the same cell in the right image.
This image was kindly supplied as part of the review submitted by Christian Schoefer.
References for Anti-Histone H3 (di+tri methyl K4) antibody [mAbcam 6000] - ChIP Grade (ab6000)
This product has been referenced in:
- Strukov YGet al. Evidence of activity-specific, radial organization of mitotic chromosomes in Drosophila. PLoS Biol 9:e1000574 (2011). ICC/IF; Fruit fly (Drosophila melanogaster).Read more (PubMed: 21264350) »
- Nakata Yet al. c-Myb, Menin, GATA-3, and MLL form a dynamic transcription complex that plays a pivotal role in human T helper type 2 cell development. Blood 116:1280-90 (2010). ChIP; Human.Read more (PubMed: 20484083) »
See all 9 publications for this product
Publishing research using ab6000? Please let us know so that we can cite the reference in this datasheet
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