Anti-Histone H3 (tri methyl K9) 抗体 - ChIP Grade (ab8898)

製品の概要

  • 製品名
    Anti-Histone H3 (tri methyl K9) antibody - ChIP Grade
    Histone H3 一次抗体 製品一覧
  • 製品の詳細
    Rabbit polyclonal to Histone H3 (tri methyl K9) - ChIP Grade
  • 由来種
    Rabbit
  • 特異性
    Specific for Histone H3 tri methyl Lysine 9. Shows slight cross-reactivity with tri methyl K27, which shares a similar epitope (please see Western blot image). Does not react with mono or di methylated K9.
  • アプリケーション
    適用あり: IHC-Fr, IHC-P, ICC/IF, ChIP, WB, ChIP/Chip, Flow Cyt, CHIPseqmore details
  • 種交差性
    交差種: Mouse, Rat, Chicken, Human, Saccharomyces cerevisiae, Xenopus laevis, Drosophila melanogaster, Indian muntjac, Xenopus tropicalis, Cyanidioschyzon merolae
    交差が予測される動物種: Mammals
  • 免疫原

    Synthetic peptide within Human Histone H3 aa 1-100 (N terminal) (tri methyl K9) conjugated to Keyhole Limpet Haemocyanin (KLH). The exact sequence is proprietary.
    (Peptide available as ab1773)

  • ポジティブ・コントロール
    • WB: Calf thymus histone preparation and HeLa whole cell extract. IHC-P: Normal human colon tissue.
  • 特記事項
    Every new batch of this antibody is tested in house in ChIP.

製品の特性

  • 製品の状態
    Liquid
  • 保存方法
    Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C or -80°C. Avoid freeze / thaw cycle.
  • バッファー
    pH: 7.40
    Preservative: 0.02% Sodium azide
    Constituent: PBS

    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...
  • 精製度
    Immunogen affinity purified
  • ポリ/モノ
    ポリクローナル
  • アイソタイプ
    IgG
  • 研究分野

アプリケーション

Our Abpromise guarantee covers the use of ab8898 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

アプリケーション Abreviews 特記事項
IHC-Fr 1/50.
IHC-P 1/400. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
ICC/IF 1/500.
ChIP Use 2-4 µg for 25 µg of chromatin.
WB Use a concentration of 1 µg/ml. Detects a band of approximately 17 kDa (predicted molecular weight: 15.4 kDa).Can be blocked with Human Histone H3 (tri methyl K9) peptide (ab1773).
ChIP/Chip Use at an assay dependent concentration.
Flow Cyt 1/100. PubMed: 24713002

ab171870 - Rabbit polyclonal IgG, is suitable for use as an isotype control with this antibody.

CHIPseq Use at an assay dependent concentration. PubMed: 21795385

ターゲット情報

  • 機能
    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.
  • 配列類似性
    Belongs to the histone H3 family.
  • 発生段階
    Expressed during S phase, then expression strongly decreases as cell division slows down during the process of differentiation.
  • 翻訳後修飾
    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.
  • 細胞内局在
    Nucleus. Chromosome.
  • Information by UniProt
  • 参照データベース
    see all
  • 別名
    • HIST1 cluster, H3J antibody
    • Histone gene cluster 1, H3 histone family, member E antibody
    • Histone gene cluster 1, H3G antibody
    • H3 histone family, member J antibody
    • HIST1 cluster, H3E antibody
    • HIST1 cluster, H3I antibody
    • Histone gene cluster 1, H3C antibody
    • FLJ92264 antibody
    • H3 histone family, member A antibody
    • H3 histone family, member B antibody
    • H3 histone family, member C antibody
    • H3 histone family, member D antibody
    • H3 histone family, member F antibody
    • H3 histone family, member H antibody
    • H3 histone family, member I antibody
    • H3 histone family, member K antibody
    • H3 histone family, member L antibody
    • H3 histone family, member T antibody
    • H3 histone, family 3A antibody
    • H3.1 antibody
    • H3.3A antibody
    • H3/a antibody
    • H3/b antibody
    • H3/c antibody
    • H3/d antibody
    • h3/f antibody
    • H3/h antibody
    • H3/i antibody
    • H3/j antibody
    • H3/k antibody
    • H3/l antibody
    • H3/t antibody
    • H31_HUMAN antibody
    • H3F1K antibody
    • H3F3 antibody
    • H3F3A antibody
    • H3FA antibody
    • H3FB antibody
    • H3FC antibody
    • H3FD antibody
    • H3FF antibody
    • H3FH antibody
    • H3FI antibody
    • H3FJ antibody
    • H3FK antibody
    • H3FL antibody
    • HIST1 cluster, H3A antibody
    • HIST1 cluster, H3B antibody
    • HIST1 cluster, H3C antibody
    • HIST1 cluster, H3D antibody
    • HIST1 cluster, H3F antibody
    • HIST1 cluster, H3G antibody
    • HIST1 cluster, H3H antibody
    • HIST1H3A antibody
    • HIST1H3B antibody
    • HIST1H3C antibody
    • HIST1H3D antibody
    • HIST1H3E antibody
    • HIST1H3F antibody
    • HIST1H3G antibody
    • HIST1H3H antibody
    • HIST1H3I antibody
    • HIST1H3J antibody
    • HIST3H3 antibody
    • Histone 1, H3a antibody
    • Histone 1, H3b antibody
    • Histone 1, H3c antibody
    • Histone 1, H3d antibody
    • Histone 1, H3e antibody
    • Histone 1, H3f antibody
    • Histone 1, H3g antibody
    • Histone 1, H3h antibody
    • Histone 1, H3i antibody
    • Histone 1, H3j antibody
    • histone 3, H3 antibody
    • histone cluster 1 H3 family member a antibody
    • histone cluster 1 H3 family member b antibody
    • histone cluster 1 H3 family member c antibody
    • histone cluster 1 H3 family member d antibody
    • histone cluster 1 H3 family member e antibody
    • histone cluster 1 H3 family member f antibody
    • histone cluster 1 H3 family member g antibody
    • histone cluster 1 H3 family member h antibody
    • histone cluster 1 H3 family member i antibody
    • histone cluster 1 H3 family member j antibody
    • Histone cluster 1, H3a antibody
    • Histone cluster 1, H3b antibody
    • Histone cluster 1, H3c antibody
    • Histone cluster 1, H3d antibody
    • Histone cluster 1, H3e antibody
    • Histone cluster 1, H3f antibody
    • Histone cluster 1, H3g antibody
    • Histone cluster 1, H3i antibody
    • Histone cluster 1, H3j antibody
    • Histone gene cluster 1, H3 histone family, member A antibody
    • Histone gene cluster 1, H3 histone family, member B antibody
    • Histone gene cluster 1, H3 histone family, member C antibody
    • Histone gene cluster 1, H3 histone family, member D antibody
    • Histone gene cluster 1, H3 histone family, member F antibody
    • Histone gene cluster 1, H3 histone family, member G antibody
    • Histone gene cluster 1, H3 histone family, member H antibody
    • Histone gene cluster 1, H3 histone family, member I antibody
    • Histone gene cluster 1, H3 histone family, member J antibody
    • Histone gene cluster 1, H3A antibody
    • Histone gene cluster 1, H3B antibody
    • Histone gene cluster 1, H3D antibody
    • Histone gene cluster 1, H3E antibody
    • Histone gene cluster 1, H3F antibody
    • Histone gene cluster 1, H3H antibody
    • Histone gene cluster 1, H3I antibody
    • Histone gene cluster 1, H3J antibody
    • Histone H 3 antibody
    • Histone H3.1 antibody
    • histone H3.1t antibody
    • Histone H3.2 antibody
    • Histone H3/a antibody
    • Histone H3/b antibody
    • Histone H3/c antibody
    • Histone H3/d antibody
    • Histone H3/f antibody
    • Histone H3/h antibody
    • Histone H3/i antibody
    • Histone H3/j antibody
    • Histone H3/k antibody
    • Histone H3/l antibody
    • Histone H3/m antibody
    • Histone H3/o antibody
    see all

画像

  • 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  ab8898 (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 for active and inactive loci, Sybr green approach for heterochromatic loci). Primers and probes are located in the first kb of the transcribed region.

  • X-Chip assay was performed using nuclear lysates prepared from mouse ES cells. Crosslinking was done for 15 minutes in 1% formaldehyde. Primary antibody was incubated first with peptides ab7228, ab1342, ab1782, ab1773, ab1772 and ab1771 in a chip competition assay and then used in chip at 0.0133µg/ µg chromatin (chip sonication buffer) and incubated with sample for 24 hours at 4°C.

    Positive control: ChIP coupled with a peptide competition assay to validate the specificity of the antibody.

    Negative control: Genomic region (chr10:79154149-79155200) with no evidence of H3K9me3. 

    RT-PCR detection method was used.

    Polrmt: PCR primers situated in the coding regions of Polymerase (RNA) mitochondrial (DNA directed).

    Agrn: PCR primers situated in the coding regions of Agrin

  • IHC image of ab8898 staining Histone H3 (tri methyl K9) in normal human colon formalin-fixed paraffin-embedded tissue sections*, performed on a Leica Bond. 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 ab8898, 1/400 dilution, 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. No primary antibody was used in the negative control (shown on the inset).

    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.

    *Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

  • ab8898 staining H3K9me3 in mouse 3T3MEF by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with PFA, permeabilized with CSK buffer and MeOH and blocked with 5% BSA for 30 minutes at 21°C. Samples were incubated with primary antibody (1/500 in PBS +5% BSA + 01% Tween20) for 2 hours at 21°C. An undiluted Alexa Fluor® 546-conjugated Goat anti-rabbit IgG was used as the secondary antibody.

    See Abreview

  • These images were kindly submitted by Prof Bryan Turner, University of Birmingham. Undifferentiated Mouse Embryonic Stem cells or cells differentiated for 7 days were incubated with ab8898. The staining is specific for centromeric heterochromatin on metaphase chromosomes.
  • All lanes : Anti-Histone H3 (tri methyl K9) antibody - ChIP Grade (ab8898) at 1 µg/ml

    Lane 1 : Calf Thymus Histone Preparation Nuclear Lysate
    Lane 2 : Calf Thymus Histone Preparation Nuclear Lysate with Human Histone H3 (unmodified ) peptide (ab7228) at 0.5 µg/ml
    Lane 3 : Calf Thymus Histone Preparation Nuclear Lysate with Human Histone H3 (mono methyl K4) peptide (ab1340) at 0.5 µg/ml
    Lane 4 : Calf Thymus Histone Preparation Nuclear Lysate with Human Histone H3 (di methyl K4) peptide (ab7768) at 0.5 µg/ml
    Lane 5 : Calf Thymus Histone Preparation Nuclear Lysate with Human Histone H3 (tri methyl K4) peptide (ab1342) at 0.5 µg/ml
    Lane 6 : Calf Thymus Histone Preparation Nuclear Lysate with Human Histone H3 (mono methyl K9) peptide (ab1771) at 0.5 µg/ml
    Lane 7 : Calf Thymus Histone Preparation Nuclear Lysate with Human Histone H3 (di methyl K9) peptide (ab1772) at 0.5 µg/ml
    Lane 8 : Calf Thymus Histone Preparation Nuclear Lysate with Human Histone H3 (tri methyl K9) peptide (ab1773) at 0.5 µg/ml
    Lane 9 : Calf Thymus Histone Preparation Nuclear Lysate with Human Histone H3 (mono methyl K27) peptide (ab1780) at 0.5 µg/ml
    Lane 10 : Calf Thymus Histone Preparation Nuclear Lysate with Human Histone H3 (di methyl K27) peptide (ab1781) at 0.5 µg/ml
    Lane 11 : Calf Thymus Histone Preparation Nuclear Lysate with Human Histone H3 (tri methyl K27) peptide (ab1782) at 0.5 µg/ml

    Lysates/proteins at 0.5 µg per lane.

    Secondary
    All lanes : IRDye 680 Conjugated Goat Anti-Rabbit IgG (H+L) at 1/10000 dilution

    Performed under reducing conditions.

    Predicted band size: 15.4 kDa
    Observed band size: 17 kDa (why is the actual band size different from the predicted?)



    Lane 8 shows that Rabbit polyclonal to Histone H3 (tri methyl K9) is blocked by the addition of the immunizing peptide (ab1773). Cross-reactivity with Histone H3 peptide - tri methyl K27 (ab1782) is also shown in Lane 11.
  • Indian muntjac fibroblast cells stained with anti-Histone H3 tri methyl K9, ab8898, (green, left panel, deconvolution image; red, right panel, epifluorescence image).

    The centromeres are enriched in Histone H3 tri methyl K9. There are also additional bands that occur throughout the chromosomes. Note that these images are taken in situ and are imaged under conditions where distinct cytogenetic-like banding patterns have not previously been possible to visualize (e.g., several acetylated antibodies have been reported to be associated with chromosome bands but, although not homogenously distributed along in situ chromosomes, they do not generate distinct banding patterns).
  • ICC/IF image of ab8898 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 (ab8898, 0.1µg/ml) overnight at +4°C. The secondary antibody (green) was ab96899, a goat anti-rabbit DyLight® 488 (IgG; H+L) 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.

  • All lanes : Anti-Histone H3 (tri methyl K9) antibody - ChIP Grade (ab8898) at 1/1000 dilution

    Lane 1 : Fruit fly embryo tissue lysate - nuclear at 4.5 µg
    Lane 2 : Fruit fly embryo tissue lysate - nuclear at 1.5 µg
    Lane 3 : Fruit fly embryo tissue lysate - nuclear at 0.5 µg

    Secondary
    All lanes : HRP-conjugated Goat anti-rabbit IgG polyclonal at 1/2000 dilution

    Developed using the ECL technique.

    Performed under reducing conditions.

    Predicted band size: 15.4 kDa
    Observed band size: 15 kDa (why is the actual band size different from the predicted?)
    Additional bands at: 45 kDa (possible non-specific binding)


    Exposure time: 1 minute

    See Abreview

プロトコール

参考文献

This product has been referenced in:
  • Gainetdinov IV  et al. Assessment of piRNA biogenesis and function in testicular germ cell tumors and their precursor germ cell neoplasia in situ. BMC Cancer 18:20 (2018). Read more (PubMed: 29301509) »
  • Muhammad K  et al. Induction of Short NFATc1/aA Isoform Interferes with Peripheral B Cell Differentiation. Front Immunol 9:32 (2018). Read more (PubMed: 29416540) »

See all 825 Publications for this product

レビューと Q&A


For ab8898 we test on the following primers :

Heterochromatin
SATa: ab83620 (f), ab83621 (r)
SAT2: ab82342 (f), ab83619 (r)

Active
GADPH: ab83588 (f), ab83589 (r)
RPL30: ab74244 (f), ab83296 (r)

Inactive<...

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I can confirm that these antibodies would immunoprecipitate the chromatin irrespective of cell line used, off course if the histone modification is present.

We unfortunately do not offer primer samples however both abs are guaranteed for chip....

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There are a number of other loading controls that can be used in WB such as TATA binding protein (ab70009) and GAPDH (ab37168).

For detecting nuclear proteins via western blot we recommend using a RIPA buffer or cell fractionation protocol in preparation of your cell lysate. Below are possible fractionation kits:
Nuclear Extraction Kit (ab113474) https://www.abcam.com/epi...

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There is a long list of references on the online datasheet, including some very recent ones. It is difficult to know which lots were used for publication, and it is likely that those lots are no longer available. In general, every lot we offer is valid...

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Unfortunately, we have not yet tested this product in ChIP on zebrafish.

The secondary antibodies you mentioned might work for dot blots with the rabbit polyclonal Histone primaries, as they are guaranteed to work in Western blot which is quite similar to dot blot. However, we have not tested them specifically for this appl...

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I assume you want to use this antibody in WB? If this is the case, I would suggest to use 3% BSA in TBST as this is what we use in our lab to establish antibodies.

The following genes can be used for control experiments:

- H3 - K9 - ab8898 product datasheet has a very information ChIP image

Positive control: Sat2 or SATa genes

Negative control: GAPDH or RPL30



- H...

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I am sorry to confirm that after checking with our laboratories, we are unable to disclose our primer sequences for our positive (or negative) control for these particular batch tested ChIP antibodies for commercial reasons. 

In this case, I...

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1-10 of 21 Q&A

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