RabMAb

Anti-Histone H3 (di methyl K4)抗体[Y47] - ChIP Grade (ab32356)

概述

  • 产品名称Anti-Histone H3 (di methyl K4)抗体[Y47] - ChIP Grade
    参阅全部 Histone H3 一抗
  • 描述
    兔单克隆抗体[Y47] to Histone H3 (di methyl K4) - ChIP Grade
  • 特异性This antibody only detects Histone H3 dimethylated on Lysine 4.
  • 经测试应用适用于: CHIPseq, IHC-Fr, ChIP, WB, IHC-P, ICC/IF, IP, Flow Cytmore details
  • 种属反应性
    与反应: Mouse, Rat, Chicken, Cow, Human, Monkey, Rice
    预测可用于: Sheep, Saccharomyces cerevisiae, Xenopus laevis, Arabidopsis thaliana, Zebrafish, all Mammals
  • 免疫原

    Synthetic peptide (the amino acid sequence is considered to be commercially sensitive) within Human Histone H3 aa 1-100 (di methyl K4). The exact sequence is proprietary.

  • 阳性对照
    • WB: HeLa, HEK-293, SH-SY5Y, C6 L6, NIH/3T3, COS-1, UMNSAH/DF-1 and MDBK cell lysates. ICC/IF: HepG2 cells. Flow Cyt: HeLa cells. IHC-P: Human cervical carcinoma, mouse colon and rat spleen tissues.
  • 常规说明

    A trial size is available to purchase for this antibody.

    Alternative versions available:
    Anti-Histone H3 (di methyl K4) antibody (Alexa Fluor® 488) [Y47] - (ab200024)
    Anti-Histone H3 (di methyl K4) antibody (Alexa Fluor® 647) [Y47] (ab200026)
    Anti-Histone H3 (di methyl K4) antibody (Alexa Fluor® 594) -  [Y47] (ab207864)
    Anti-Histone H3 (di methyl K4) antibody - (Phycoerythrin) [Y47] (ab208741)

    Every new batch of this antibody is tested in house in ChIP.

    Produced using Abcam's RabMAb® technology. RabMAb® technology is covered by the following U.S. Patents, No. 5, 675, 063 and/or 7, 429, 487.

    We are constantly working hard to ensure we provide our customers with best in class antibodies. As a result of this work we are pleased to now offer this antibody in purified format. We are in the process of updating our datasheets. The purified format is designated 'PUR' on our product labels. If you have any questions regarding this update, please contact our Scientific Support team.

    This product is a recombinant rabbit monoclonal antibody.

性能

应用

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.

应用 Ab评论 说明
CHIPseq Use at an assay dependent concentration. PubMed: 19581485
IHC-Fr Use at an assay dependent concentration.
ChIP Use 2 µg for 25 µg of chromatin.
WB 1/2000. Detects a band of approximately 17 kDa (predicted molecular weight: 15 kDa).
IHC-P 1/800.
ICC/IF 1/1000.
IP Use at an assay dependent concentration. PubMed: 22086061
Flow Cyt 1/20 - 1/100.

ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.

靶标

  • 功能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.
  • 序列相似性Belongs to the histone H3 family.
  • 发展阶段Expressed during S phase, then expression strongly decreases as cell division slows down during the process of differentiation.
  • 翻译后修饰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.
  • 细胞定位Nucleus. Chromosome.
  • Information by UniProt
  • 数据库链接
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  • 别名
    • 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

Anti-Histone H3 (di methyl K4) antibody [Y47] - ChIP Grade 图像

  • All lanes : Anti-Histone H3 (di methyl K4) antibody [Y47] - ChIP Grade (ab32356) at 1/10000 dilution (purified)

    Lane 1 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate
    Lane 2 : HEK-293 (Human epithelial cell line from embryonic kidney) whole cell lysate
    Lane 3 : SH-SY5Y (Human neuroblastoma cell line from bone marrow) whole cell lysate
    Lane 4 : C6 (Rat glial tumor cell line) whole cell lysate

    Lysates/proteins at 20 µg per lane.

    Secondary
    Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

    Predicted band size : 15 kDa
    Additional bands at : 17 kDa. We are unsure as to the identity of these extra bands.

    Exposure time : 5 seconds
  • Chromatin was prepared from HeLa cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10 minutes. The ChIP was performed with 25µg of chromatin, 2µg of ab32356 (blue), and 20µl of Anti Rabbit IgG sepharose beads. 2μg of rabbit normal IgG was added to the beads control (yellow). The immunoprecipitated DNA was quantified by real time PCR (Sybr green approach). Primers and probes are located in the first kb of the transcribed region.

  • All lanes : Anti-Histone H3 (di methyl K4) antibody [Y47] - ChIP Grade (ab32356) at 1/2000 dilution (purified)

    Lane 1 : L6 (Rat skeletal muscle cell line) whole cell lysate
    Lane 2 : NIH/3T3 (Mouse embryonic fibroblast cell line) whole cell lysate
    Lane 3 : COS-1 (African green monkey kidney fibroblast-like cell line) whole cell lysate
    Lane 4 : UMNSAH/DF-1 (Transformed chicken embryonic fibroblast cell line) whole cell lysate
    Lane 5 : MDBK (Bovine kidney cell line) whole cell lysate

    Lysates/proteins at 20 µg per lane.

    Secondary
    Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/1000 dilution

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


    Exposure time : 5 seconds
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human cervical carcinoma tissue labelling Histone H3 (di methyl K4) with purified ab32356 at a dilution of 1/800. Antigen retrieval was performed using Tris/EDTA buffer, pH9. ab97051, a HRP-conjugated goat anti-rabbit IgG H&L was used as the secondary antibody (1/500). Counter stained with hematoxylin.

  • Immunocytochemistry/Immunofluorescence analysis of HepG2 cells labelling Histone H3 (di methyl K4) with purified ab32356 at a dilution of 1/1000. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. ab150077, an Alexa Fluor® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody. The cells were co-stained with ab7291, a mouse anti-tubulin (1/1000) using ab150120, an Alexa Fluor® 594-conjugated goat anti-mouse IgG (1/1000) as the secondary antibody. Nuclei counterstained with DAPI (blue). 

    Control 1: primary antibody (1/1000) and secondary antibody, ab150120, an Alexa Fluor® 594-conjugated goat anti-mouse IgG (1/1000).

    Control 2: ab7291 (1/1000) and secondary antibody, ab150077, an Alexa Fluor® 488-conjugated goat anti-rabbit IgG (1/1000).

  • Flow Cytometry analysis of HeLa cells labelling Histone H3 (di methyl K4) with purified ab32356 at 1/150 (red). Cells were fixed with 80% methanol. An Alexa Fluor® 488-conjugated goat anti-rabbit IgG (1/500) was used as the secondary antibody. Black - Isotype control, rabbit monoclonal IgG (ab172730). Blue - Unlabelled control, cells without incubation with primary and secondary antibodies.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of mouse colon tissue labelling Histone H3 (di methyl K4) with purified ab32356 at a dilution of 1/800. Antigen retrieval was performed using Tris/EDTA buffer, pH9. ab97051, a HRP-conjugated goat anti-rabbit IgG H&L was used as the secondary antibody (1/500). Counter stained with hematoxylin.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of rat spleen tissue labelling Histone H3 (di methyl K4) with purified ab32356 at a dilution of 1/800. Antigen retrieval was performed using Tris/EDTA buffer, pH9. ab97051, a HRP-conjugated goat anti-rabbit IgG H&L was used as the secondary antibody (1/500). Counter stained with hematoxylin.

  •     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. Every new batch of this antibody is tested at Abcam in ChIP.

     

  • Unpurified 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.

    See Abreview

  • ICC/IF image of unpurified 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 a goat anti-rabbit DyLight® 488 (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 unpurified 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 a goat anti-rabbit DyLight® 488 (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 (unpurified)

    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?)

Anti-Histone H3 (di methyl K4) antibody [Y47] - ChIP Grade (ab32356)参考文献

This product has been referenced in:
  • Xiong Y  et al. Inhibition of Lysine-Specific Demethylase-1 (LSD1/KDM1A) Promotes the Adipogenic Differentiation of hESCs Through H3K4 Methylation. Stem Cell Rev N/A:N/A (2016). WB . Read more (PubMed: 27059868) »
  • Velinder M  et al. GFI1 functions in transcriptional control and cell fate determination require SNAG domain methylation to recruit LSD1. Biochem J 473:3355-69 (2016). Read more (PubMed: 27480105) »

See all 39 Publications for this product

Product Wall

Abcam guarantees this product to work in the species/application used in this Abreview.
Application Immunocytochemistry/ Immunofluorescence
Sample Mouse Cell (NIH3T3)
Specification NIH3T3
Fixative Paraformaldehyde
Permeabilization Yes - 0.1% Triton
Blocking step Serum as blocking agent for 30 minute(s) · Concentration: 10% · Temperature: 24°C
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Verified customer

提交于 Mar 22 2013

Abcam has not validated the combination of species/application used in this Abreview.
Application Western blot
Sample Fruit fly (Drosophila melanogaster) Tissue lysate - whole (Whole fly tissue)
Gel Running Conditions Reduced Denaturing (gradient precasted gel, Biorad 567-8124)
Loading amount 40 µg
Specification Whole fly tissue
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
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提交于 May 06 2016


For ab32356 (http://www.abcam.com/histone-h3-di-methyl-k4-antibody-y47-chip-grade-ab32356.html):
Based on our western blot testing testing, ab32356 should specifically detect Histone H3 dimethylated on Lysine 4. The WB figure on the product da...

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Abcam guarantees this product to work in the species/application used in this Abreview.
Application Immunohistochemistry (Frozen sections)
Sample Rat Tissue sections (Rat airway epithelium and alveoli)
Specification Rat airway epithelium and alveoli
Fixative Paraformaldehyde
Permeabilization Yes - 0.1% Triton
Blocking step BSA as blocking agent for 30 minute(s) · Concentration: 1% · Temperature: 27°C
Username

Abcam user community

Verified customer

提交于 Nov 15 2012

Abcam guarantees this product to work in the species/application used in this Abreview.
Application Immunohistochemistry (Frozen sections)
Sample Human Tissue sections (Human lung endothelial lining)
Specification Human lung endothelial lining
Fixative Paraformaldehyde
Permeabilization Yes - 0.1% Triton
Blocking step BSA as blocking agent for 30 minute(s) · Concentration: 1% · Temperature: 27°C
Username

Abcam user community

Verified customer

提交于 Nov 15 2012

Abcam guarantees this product to work in the species/application used in this Abreview.
Application Immunohistochemistry (Frozen sections)
Sample Mouse Tissue sections (Mouse bronchiolar epithelium)
Specification Mouse bronchiolar epithelium
Fixative Paraformaldehyde
Permeabilization Yes - 0.1% Triton
Blocking step BSA as blocking agent for 30 minute(s) · Concentration: 1% · Temperature: 27°C
Username

Abcam user community

Verified customer

提交于 Nov 15 2012

Abcam guarantees this product to work in the species/application used in this Abreview.
Application Immunocytochemistry/ Immunofluorescence
Sample Monkey Cell (COS1)
Specification COS1
Fixative Paraformaldehyde
Permeabilization Yes - 0.1% Triton X-100
Blocking step Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: 25°C
Username

Abcam user community

Verified customer

提交于 Nov 06 2012

Abcam guarantees this product to work in the species/application used in this Abreview.
Application Western blot
Sample Monkey Cell lysate - whole cell (COS1)
Loading amount 20 µg
Specification COS1
Gel Running Conditions Reduced Denaturing (12%)
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
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Abcam user community

Verified customer

提交于 Nov 05 2012

While the IgG produced by rabbits have greater affinity and greater sensitivity than mouse IgG (about 100-1000 fold), the total amount of IgG produced by the rabbit hybridomas is generally lower than mice. Since many people are not aware of this fact p...

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