Anti-Histone H3 (tri methyl K4) 抗体 [mAbcam12209] - ChIP Grade (ab12209)

製品の概要

  • 製品名

    Anti-Histone H3 (tri methyl K4) antibody [mAbcam12209] - ChIP Grade
    Histone H3 一次抗体 製品一覧
  • 製品の詳細

    Mouse monoclonal [mAbcam12209] to Histone H3 (tri methyl K4) - ChIP Grade
  • 由来種

    Mouse
  • 特異性

    ab12209 is strongly blocked in Western blotting on histones by tri methyl K4, weakly by di methyl K4 and very weakly by mono methyl K4 peptides. It is not blocked by non-modified peptides. By ELISA the antibody binds to the tri methyl K4 peptide and at high antibody concentrations to di and mono methyl K4 peptides. It does not bind to unmodified, mono, di or tri methyl K9 or di or tri methyl K27 peptides. Not suitable for blocking with milk in Western blot (see Application notes).
  • アプリケーション

    適用あり: Flow Cyt, WB, ICC/IF, ChIP, ELISA, IPmore details
  • 種交差性

    交差種: Mouse, Rat, Human
    交差が予測される動物種: Saccharomyces cerevisiae, Xenopus laevis, Arabidopsis thaliana, Caenorhabditis elegans, Drosophila melanogaster, Schizosaccharomyces pombe, Zebrafish, Mammals, Neurospora crassa
  • 免疫原

    Synthetic peptide within Human Histone H3 aa 1-100 (tri methyl K4) conjugated to keyhole limpet haemocyanin. The exact sequence is proprietary.
    (Peptide available as ab1342)

  • ポジティブ・コントロール

    • FC: HeLa cells; ICC/IF: HeLa cells; WB: Calf Thymus Histone Preparation Nuclear Lysate; ChIP: U2OS cells.
  • 特記事項

    This antibody clone is manufactured by Abcam.

    If you require this antibody in a particular buffer formulation or a particular conjugate for your experiments, please contact orders@abcam.com or you can find further information here.

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
    Constituents: PBS, 6.97% L-Arginine
  • Concentration information loading...
  • 精製度

    IgG fraction
  • ポリ/モノ

    モノクローナル
  • クローン名

    mAbcam12209
  • ミエローマ

    Sp2/0-Ag14
  • アイソタイプ

    IgG1
  • 軽鎖の種類

    kappa
  • 研究分野

アプリケーション

Our Abpromise guarantee covers the use of ab12209 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
Flow Cyt Use 1µg for 106 cells.

ab170190 - Mouse monoclonal IgG1, is suitable for use as an isotype control with this antibody.

 

WB Use a concentration of 1 - 5 µg/ml. Detects a band of approximately 17 kDa (predicted molecular weight: 15 kDa).Can be blocked with Human Histone H3 (tri methyl K4) peptide (ab1342). NOT SUITABLE for blocking with milk. Block in 5% BSA for 1 hour. Our labs have investigated the blocking conditions for this antibody and found that milk significantly decreases the signal and is therefore not a suitable blocking agent for this antibody (see Western Blot image).
ICC/IF Use a concentration of 5 µg/ml.
ChIP Use 2-5 µg for 25 µg of chromatin.
ELISA Use at an assay dependent concentration.
IP Use at an assay dependent concentration. PubMed: 22086061

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

  • 別名

    • H3 histone family member E pseudogene antibody
    • H3 histone family, member A antibody
    • H3/A antibody
    • H31_HUMAN antibody
    • H3F3 antibody
    • H3FA 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 cluster 1, H3a antibody
    • Histone H3 3 pseudogene antibody
    • Histone H3.1 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
    see all

画像

  • 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 ab12209 (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.

  • Ab12209 staining Histone H3 (Tri Methyl K4) in Mouse hair follicle DSCs by ICC/IF (Immunocytochemistry/Immunofluorescence).  Cells were fixed with 3.7% paraformaldehyde, permeabilised with 0.2% Triton X-100 and blocked with 10% normal goat serum in PBS.  Samples were incubated with primary antibody at 1:200 dilution. An Alexa Fluor ® 568 conjugated goat anti-mouse IgG was used as a secondary antibody.

  • All lanes : Anti-Histone H3 (tri methyl K4) antibody [mAbcam12209] - ChIP Grade (ab12209) at 2 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 0.25 µg/ml
    Lane 3 : Calf Thymus Histone Preparation Nuclear Lysate with Human Histone H3 (mono methyl K4) peptide (ab1340) at 0.25 µg/ml
    Lane 4 : Calf Thymus Histone Preparation Nuclear Lysate with Human Histone H3 (di methyl K4) peptide (ab7768) at 0.25 µg/ml
    Lane 5 : Calf Thymus Histone Preparation Nuclear Lysate with Human Histone H3 (tri methyl K4) peptide (ab1342) at 0.25 µg/ml
    Lane 6 : Calf Thymus Histone Preparation Nuclear Lysate with Human Histone H3 (mono methyl K9) peptide (ab1771) at 0.25 µg/ml
    Lane 7 : Calf Thymus Histone Preparation Nuclear Lysate with Human Histone H3 (di methyl K9) peptide (ab1772) at 0.25 µg/ml
    Lane 8 : Calf Thymus Histone Preparation Nuclear Lysate with Human Histone H3 (tri methyl K9) peptide (ab1773) at 0.25 µg/ml
    Lane 9 : Calf Thymus Histone Preparation Nuclear Lysate with Human Histone H3 (mono methyl K27) peptide (ab1780) at 0.25 µg/ml
    Lane 10 : Calf Thymus Histone Preparation Nuclear Lysate with Human Histone H3 (di methyl K27) peptide (ab1781) at 0.25 µg/ml
    Lane 11 : Calf Thymus Histone Preparation Nuclear Lysate with Human Histone H3 (tri methyl K27) peptide (ab1782) at 0.25 µg/ml

    Lysates/proteins at 0.5 µg per lane.

    Secondary
    All lanes : Goat polyclonal to Mouse IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

    Performed under reducing conditions.

    Predicted band size: 15 kDa
    Observed band size: 17 kDa
    why is the actual band size different from the predicted?


    Exposure time: 16 minutes
  • ICC/IF image of ab12209 stained human HeLa cells. The cells were 4% PFA fixed (10 min), permabilised in 0.1% PBS-Tween (20 min) and incubated with the antibody (ab12209, 5µg/ml) for 1h at room temperature. 1%BSA / 10% normal goat serum / 0.3M glycine was used to block non-specific protein-protein interactions. 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). DAPI was used to stain the cell nuclei (blue).

  • ELISA using ab12209 at varying antibody concentrations.

    Curve_SPL4  indicates binding to the tri methyl K4 peptide ab1342. In addition, SPL3 indicates partial binding to the di methyl K4 peptide ab7768.  There is very weak cross-reactivity with the mono methyl K4 peptide ab1340 (Curve_SPL2).

    Binding to the following peptides was not seen:
    SPL1 unmodified Histone H3, SPL5 Histone H3 mono methyl K9, SPL6 Histone H3 di methyl K9, SPL7 Histone H3 tri methyl K9, SPL8 Histone H3 mono methyl K27, SPL9 Histone H3 di methyl K27, SPL10 Histone H3 tri methyl K27.

  • Lane 1 : Anti-Histone H3 (tri methyl K4) antibody [mAbcam12209] - ChIP Grade (ab12209) at 1 µg/ml (BLOCKED WITH 5% BSA)
    Lane 2 : Anti-Histone H3 (tri methyl K4) antibody [mAbcam12209] - ChIP Grade (ab12209) at 1 µg/ml (BLOCKED WITH 5% MILK)

    All lanes : Calf Thymus Histone Preparation Nuclear Lysate

    Lysates/proteins at 0.5 µg per lane.

    Secondary
    All lanes : Goat polyclonal to Mouse IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

    Performed under reducing conditions.

    Predicted band size: 15 kDa
    Observed band size: 17 kDa why is the actual band size different from the predicted?


    Exposure time: 12 minutes
  • Overlay histogram showing HeLa cells stained with ab12209 (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 (ab12209, 1µg/1x106 cells) for 30 min at 22ºC. The secondary antibody used was a goat anti-mouse DyLight® 488 (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.

プロトコール

参考文献

This product has been referenced in:

  • Marquez J  et al. NADH dehydrogenase complex I is overexpressed in incipient metastatic murine colon cancer cells. Oncol Rep 41:742-752 (2019). Read more (PubMed: 30483808) »
  • Bae J  et al. Phc2 controls hematopoietic stem and progenitor cell mobilization from bone marrow by repressing Vcam1 expression. Nat Commun 10:3496 (2019). Read more (PubMed: 31375680) »
See all 29 Publications for this product

レビューと Q&A

1-10 of 12 Abreviews or Q&A

Application
Western blot
Sample
Pig Tissue lysate - whole (Liver)
Gel Running Conditions
Reduced Denaturing (17,5)
Loading amount
20 µg
Specification
Liver
Blocking step
BSA as blocking agent for 30 minute(s) · Concentration: 3% · Temperature: 22°C

Abcam user community

Verified customer

投稿 Nov 28 2017

Application
Immunohistochemistry (Frozen sections)
Sample
Mouse Tissue sections (Skin)
Permeabilization
No
Specification
Skin
Blocking step
Serum as blocking agent for 30 minute(s) · Concentration: 10% · Temperature: 25°C
Fixative
Paraformaldehyde

Ahmar Aziz

Verified customer

投稿 Feb 09 2016

Answer

I can confirm that Histone H3 (tri methyl K4) antibody [mAbcam12209] ab12209 is currently provided without BSA and glycerol. Any additional buffer constituents such as these would be listed on the datasheet in the buffer and preservative section.

Read More

Answer

>We currently do not offer ab8580 in a buffer which is free of BSA. When using our EasyLink Conjugation Kits concentrations of up to 0.5% BSA and 0.1% gelatin have little effect on the conjugation chemistry. As such I would suggest one of the following options.

1) Purify the antibody to remove the BSA and Sodium Azide. We carry antibody purification kits which canquickly remove these contaminants. Our Protein G purification kit may be found here:https://www.abcam.com/Antibody-Purification-Kit-Protein-G-ab128747.html

2) Use another anti Histone H3 K4me3 antibody which does not contain BSA or other additive at levels which will interfere with your conjugation.AB12209 contains only 0.2% Sodium Azide and no BSA.https://www.abcam.com/Histone-H3-tri-methyl-K4-antibody-mAbcam12209-ChIP-Grade-ab12209.html

More information and FAQs about EasyLink Conjugation Kits may be found at these links:

FAQs: https://www.abcam.com/index.html?pageconfig=resource&rid=13156

EasyLink:https://www.abcam.com/index.html?pageconfig=resource&rid=13148

We are currently running a promotion through August for 25% off of any EasyLink conjugation kit by using the codeNEW-F76ZBby 31 August 2012.

As always, these products are covered by our Abpromise guarantee. We provide scientific support, replacement or refund should this product not perform as indicated on the datasheet. More information on our Abpromise may be found at the following link:

https://www.abcam.com/index.html?pageconfig=abpromise

Read More

Answer

These antibodies are all purified antibodies. Note though, that we do not test for RNAse cactivity.
The ab6002 is a monoclonal antibody IgG fraction, in Sodium Chloride solution containing Azide.
The ab9050 is a monoclonal antibody purified by affinity to the immunogen (on a column with immobilized immunogen peptide), in a PBS buffer, containing Azide and cArginine (for stability).
The ab12209 is a monoclonal antibody IgG fraction as well in PBS buffer with azide.

Read More

Answer

I can confirm that H3K27me3 antibody (ab6002) currently in stock has a concentration of 0.6mg/ml and that we recommend to use 4µg for 106 cells in CHIP. We also offer a H3K36me3 antibody tested and guaranteed for ChIP: ab9050 https://www.abcam.com/index.html?datasheet=9050 https://www.abcam.com/index.html?datasheet=9050. The lots currently in stock have a concentration of 1mg/ml and we recommend to use 5-10 µg for 25 µg of chromatin. The optimal concentration for individual experiments always has to be determined by the users themselves. None of these antibodies (ab6002, ab9050, ab12209) is tested and guaranteed for RIP. Since they all work in CHIP, it is very likely they will also be suitable for RIP. Therefore, I can offer a discount off a future purchase if you buy ab6002, ab9050 and/or ab12209 now, test it in RIP and submit feedback to using our AbTrial program. For more information or to enroll in the AbTrial testing program doe untested applications and species please let me know.

Read More
Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (HeLa)
Specification
HeLa
Fixative
Paraformaldehyde
Permeabilization
Yes - 0.5% Triton X100

Dr. Kirk Mcmanus

Verified customer

投稿 Jun 07 2011

Application
Western blot
Sample
Mouse Purified protein (kidney, acid-extracted histone)
Loading amount
10 µg
Specification
kidney, acid-extracted histone
Gel Running Conditions
Reduced Denaturing
Blocking step
Milk as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 24°C

Abcam user community

Verified customer

投稿 Apr 08 2011

Application
ChIP
Sample
Arabidopsis thaliana Tissue lysate - nuclear (seedling, 300 mg fresh weight.)
Specification
seedling, 300 mg fresh weight.
Type
Native ChIP (N-ChIP)
Detection step
Real-time PCR
Positive control
I used antibodies that I know work as a positive control for the whole experiment.
qPCR for positive controls used in various publications, e.g. 5' regions of housekeeping genes (actin, At4g04040, both of which are active in my tissue).
Negative control
qPCR for At4g03770 and At4g03880 (silenced transposons)

Abcam user community

Verified customer

投稿 Feb 02 2011

1-10 of 12 Abreviews or Q&A

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