By peptide ELISA ab1220 recognizes di methyl K9, but not unmodified K9, mono methyl K9, tri methyl K9, di methyl K27, tri methyl K27, mono methyl K4, di methyl K4 or tri methyl K4. By Western blot ab1220 is blocked by di methyl K9, but not by unmodified K9, mono methyl K9, tri methyl K9, di methyl K27, tri methyl K27, mono methyl K4, di methyl K4 or tri methyl K4. This indicates the specificity of ab1220 for di methyl K9 of Histone H3.
Mouse, Rat, Chicken, Cow, Human, Xenopus laevis, Arabidopsis thaliana, Caenorhabditis elegans, Drosophila melanogaster, Schizosaccharomyces pombe, Corn, Common marmoset, Rice, Other 交差が予測される動物種:
Sheep, Saccharomyces cerevisiae
Synthetic peptide corresponding to Human Histone H3 aa 1-100 (di methyl K9) conjugated to keyhole limpet haemocyanin (Cysteine residue). Clones were positively screened by ELISA against the immunising peptide. Clones were negatively screened against both the non-modified equivalent peptide and against a dimethylated K27 peptide. (Peptide available as ab1772)
IHC-P: Human kidney tissue; Rat liver tissue. ICC/IF: HeLa cells. Flow Cytometry: Mouse embryonic stem cells. WB: HeLa whole cell lysate.
ab170191 - Mouse monoclonal IgG2a, is suitable for use as an isotype control with this antibody.
Use at an assay dependent concentration.
Use a concentration of 1 - 0.00025 µg/ml. when testing with immunogen peptide.
Use at an assay dependent concentration.
Use 2-4 µg for 25 µg of chromatin.
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.
Belongs to the histone H3 family.
Expressed during S phase, then expression strongly decreases as cell division slows down during the process of differentiation.
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.
Lane 1 : Calf thymus histone lysate Lane 2 : Calf thymus histone lysate with Histone H3 peptide - unmodified at 1 µg/ml Lane 3 : Calf thymus histone lysate with Human Histone H3 (mono methyl K9) peptide (ab1771) at 1 µg/ml Lane 4 : Calf thymus histone lysate with Human Histone H3 (di methyl K9) peptide (ab1772) at 1 µg/ml Lane 5 : Calf thymus histone lysate with Human Histone H3 (tri methyl K9) peptide (ab1773) at 1 µg/ml Lane 6 : Calf thymus histone lysate with Human Histone H3 (di methyl K4) peptide (ab7768) at 1 µg/ml Lane 7 : Calf thymus histone lysate with Human Histone H3 (di methyl K27) peptide (ab1781) at 1 µg/ml
Secondary Rabbit Anti-Mouse IgG H&L (HRP) (ab6728) at 1/5000 dilution Developed using the ECL technique
Performed under reducing conditions.
Predicted band size : 17 kDa
Exposure time : 1 minute
Immunocytochemistry/ Immunofluorescence - Anti-Histone H3 (di methyl K9) antibody [mAbcam 1220] - ChIP Grade (ab1220)This image is courtesy of Mr Mark Rochman by Abreview.
ab1220 staining Histone H3 (di methyl K9) in MEFs SV40 transformed by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with PFA, permeabilized with 0.2% Triton X-100 in PBS and blocked with 3% BSA for 1 hour at 21°C. Samples were incubated with primary antibody (1/200) for 1 hour at 21°C. A Cy3® conjugated Goat anti-mouse was used as the secondary antibody.
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 25µg of chromatin, 2µg of ab1220 (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 for active and inactive loci, Sybr green approach for heterochromatic loci). Primers and probes are located in the first Kb of the transcribed region.
ab1220 staining human kidney sections by IHC-P using EXPOSE IHC detection kit (ab80436). Formalin fixed paraffin embedded tissue sections were pre-treated using heat mediated antigen retrieval (using a pressure cooker) with sodium citrate buffer (pH6) for 30 mins. The section was incubated with ab1220, 5µg/ml, for 1 hour at room temperature. DAB was used as the chromogen and the section was counterstained with haematoxylin and mounted with DPX.
ELISA using ab1220 at varying antibody concentrations.
The purple line indicates binding to the di methyl K9 peptide ab1772. Binding to the following peptides was not seen: unmodified K9 (ab2903), mono methyl K9 (ab1771), tri methyl K9 (ab1773), di methyl K27 (ab1781), tri methyl K27 peptide (ab1782), mono methyl K4 (ab1340), di methyl K4 (ab7768), tri methyl K4 (ab1342).
This indicates the specificity of ab1220 for di methyl K9 of Histone H3.
Western blot - Anti-Histone H3 (di methyl K9) antibody [mAbcam 1220] - ChIP Grade (ab1220)
Anti-Histone H3 (di methyl K9) antibody [mAbcam 1220] - ChIP Grade (ab1220) at 1 µg/ml + HeLa (Human epitherlial carcinoma cell line) Whole Cell Lysate at 20 µg
Secondary Goat polyclonal to Mouse IgG-H&L- Pre-Adsorbed (HRP) at 1/10000 dilution Developed using the ECL technique
Performed under reducing conditions.
Predicted band size : 17 kDa Observed band size : 17 kDa
Exposure time : 5 minutes
Lane 1 : Marker.
All blocking and antibody incubation steps were done with 5% milk in 20mM Tris-HCL, and0.1% TWEEN-20.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H3 (di methyl K9) antibody [mAbcam 1220] - ChIP Grade (ab1220)This image is a courtesy of Anonymous Abreview
ab1220 staining Histone H3 (di methyl K9) in Mouse Tissue sections (Liver, P5) by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with paraformaldehyde and blocked with 5% BSA for 1 hour at 25°C; antigen retrieval was by heat mediation in a citrate buffer. Samples were incubated with primary antibody (1/100 in PBS/Triton 0,05%) for 3 hours at 25°C. An Alexa Fluor® 488 conjugated Goat anti-mouse (1/250) was used as the secondary antibody.
Flow Cytometry - Anti-Histone H3 (di methyl K9) antibody [mAbcam 1220] - ChIP Grade (ab1220)This image is courtesy of an Abreview submitted by Prof Albrecht Müller
ab1220 staining mouse embryonic stem cells by flow cytometry (gated on all living cells). The cells were trypsinized and stained with the antibody at 1ug/1.5 x 105 cells in a permeabilization buffer. A Cy3® conjugated goat anti-mouse antibody was used as the secondary.
Yang Y et al. SAC3B, a central component of the mRNA export complex TREX-2, is required for prevention of epigenetic gene silencing in Arabidopsis. Nucleic Acids Res45:181-197 (2017).
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