Anti-Histone H3 (di methyl K4) antibody [Y47] - ChIP Grade (ab32356)
- Product nameAnti-Histone H3 (di methyl K4) antibody [Y47] - ChIP GradeSee all Histone H3 (methylated) primary antibodies ...
- DescriptionRabbit monoclonal [Y47] to Histone H3 (di methyl K4) - ChIP Grade
- SpecificityThis antibody only detects Histone H3 dimethylated on Lysine 4.
- Tested applicationsChIP, CHIPseq, IHC-Fr, WB, IHC-P, ICC/IF, IP, Flow Cyt more details
- Species reactivityReacts with: Mouse, Rat, Human, Monkey
Predicted to work with: Sheep, Chicken, Cow, Saccharomyces cerevisiae, Xenopus laevis, Arabidopsis thaliana, Zebrafish, all Mammals, Rice
A synthetic (Dimethyl K) peptide corresponding to residues surrounding Lys4 of Histone H3.
- Positive controlWB: HeLa cell lysate. IF: HeLa cells. IHC-P: Breast carcinoma.
- General notesProduced under U.S. Patent No. 5,675,063.
- Storage instructionsShipped at 4°C. Upon delivery aliquot and store at -20°C. Avoid freeze / thaw cycles.
- Storage bufferPBS 49%,Sodium azide 0.01%,Glycerol 50%,BSA 0.05%
- Concentration information loading...
- Clonality Monoclonal
- Clone numberY47
- Research Areas
Our Abpromise guarantee covers the use of ab32356 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. Use 8µl for 25µg of chromatin.|
|CHIPseq||CHIPseq: Use at an assay dependent dilution. PubMed: 19581485|
|IHC-Fr||IHC-Fr: Use at an assay dependent concentration.|
|WB||WB: 1/1000 - 1/10000. Detects a band of approximately 17 kDa (predicted molecular weight: 15 kDa).|
|IHC-P||IHC-P: Use at an assay dependent dilution.|
|IP||IP: Use at an assay dependent concentration. PubMed: 22086061|
|Flow Cyt||Flow Cyt: 1/100.|
- 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: 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
- 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: P84229 Chicken
- SwissProt: P68432 Cow
- 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 (di methyl K4) antibody [Y47] - ChIP Grade images
Chromatin was prepared from Hela cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10min. The ChIP was performed with 25µg of chromatin, 8µl of ab32356 (blue), and 20µl 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.
Ab32356, at a 1/250 dilution, staining Histone H3 in HeLa cells by Immunofluorescence.
ab32356 (1/200) staining Histone H3 (di methyl K4) in HeLa cells (green). Cells were fixed in paraformaldehyde, permeabilized with 0.5% Triton X100 and counterstained with DAPI in order to highlight the nucleus. For further experimental details please refer to Abreview.
ICC/IF image of ab32356 stained HepG2 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 (ab32356 , 5µg/ml) overnight at +4°C. The secondary antibody (green) was DyLight® 488 goat anti-rabbit IgG - H&L, pre-adsorbed (ab96899) used at a 1/250 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.
Overlay histogram showing HeLa cells stained with ab32356 (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 (ab32356, 1/100 dilution) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed.
All lanes : Anti-Histone H3 (di methyl K4) antibody [Y47] - ChIP Grade (ab32356) at 1/20000 dilution
Lane 1 : HeLa cell lysate
Lane 2 : Non methylated recombinant Histone H3
Predicted band size : 15 kDa
Observed band size : 17 kDa (why is the actual band size different from the predicted?)
Ab32356, at a 1/100 dilution, staining Histone H3 in paraffin embedded breast carcinoma tissue sections by Immunohistochemistry.
References for Anti-Histone H3 (di methyl K4) antibody [Y47] - ChIP Grade (ab32356)
This product has been referenced in:
- Patel M et al. Tumor-specific retargeting of an oncogenic transcription factor chimera results in dysregulation of chromatin and transcription. Genome Res 22:259-70 (2012). IP ; Human . Read more (PubMed: 22086061) »
- Tiwari VK et al. Target genes of Topoisomerase IIß regulate neuronal survival and are defined by their chromatin state. Proc Natl Acad Sci U S A 109:E934-43 (2012). ChIP . Read more (PubMed: 22474351) »