Recombinant
RabMAb

Anti-Histone H3 (tri methyl K27) antibody [EPR18607] - ChIP Grade (ab192985)

Overview

  • Product name
    Anti-Histone H3 (tri methyl K27) antibody [EPR18607] - ChIP Grade
    See all Histone H3 primary antibodies
  • Description
    Rabbit monoclonal [EPR18607] to Histone H3 (tri methyl K27) - ChIP Grade
  • Tested applications
    Suitable for: IHC-P, ChIP, ICC/IF, WB, PepArr, ELISAmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Human
  • Immunogen

    Synthetic peptide (the amino acid sequence is considered to be commercially sensitive) within Human Histone H3 aa 1-100 (tri methyl K27). The exact sequence is proprietary.
    Database link: P68431

  • Positive control
    • WB: HeLa and NIH/3T3 whole cell lysates. IHC-P: Human colon, mouse colon and rat kidney tissues. ICC/IF: HeLa cells. ChIP: Chromatin prepared from HeLa cells. ELISA: Histone H3 – unmodified, Histone H3 - mono methyl K4, Histone H3 - di methyl K4, Histone H3 - tri methyl K4, Histone H3 - mono methyl K9, Histone H3 - di methyl K9, Histone H3 - tri methyl K9, Histone H3 - mono methyl K27, Histone H3 - di methyl K27 and Histone H3 - tri methyl K27 peptides.
  • General notes

    Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMab® patents

    This product is a recombinant rabbit monoclonal antibody.

Properties

  • Form
    Liquid
  • Storage instructions
    Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C long term. Avoid freeze / thaw cycle.
  • Storage buffer
    Preservative: 0.01% Sodium azide
    Constituents: 59% PBS, 40% Glycerol, 0.05% BSA
  • Concentration information loading...
  • Purity
    Protein A purified
  • Clonality
    Monoclonal
  • Clone number
    EPR18607
  • Isotype
    IgG
  • Research areas

Applications

Our Abpromise guarantee covers the use of ab192985 in the following tested applications.

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

Application Abreviews Notes
IHC-P 1/500. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
ChIP Use 2 µg for 25 µg of chromatin.
ICC/IF 1/1000.
WB 1/1000. Detects a band of approximately 15 kDa (predicted molecular weight: 15 kDa).
PepArr Use a concentration of 0.1 µg/ml.
ELISA Use a concentration of 0.25 µg/ml.

Target

  • Function
    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.
  • Sequence similarities
    Belongs to the histone H3 family.
  • Developmental stage
    Expressed during S phase, then expression strongly decreases as cell division slows down during the process of differentiation.
  • Post-translational
    modifications
    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.
  • Cellular localization
    Nucleus. Chromosome.
  • Information by UniProt
  • Database links
    see all
  • Alternative names
    • 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

Images

  • Chromatin was prepared from HeLa (Human epithelial cell line from cervix adenocarcinoma) 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 ab192985 (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).

  • Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (Human epithelial cells from cervix adenocarcinoma) cells labeling Histone H3 (tri methyl K27) with ab192985 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing nuclear staining on HeLa cell line. The nuclear counterstain is DAPI (blue).

    Tubulin is detected with Anti-alpha Tubulin mouse MAb (ab7291) at 1/1000 dilution, followed by Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) (ab150120) secondary antibody at 1/1000 dilution (red).

    The negative controls are as follows:-
    -ve control 1: ab192985 at 1/1000 dilution, followed by Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) (ab150120) secondary antibody at 1/1000 dilution.
    -ve control 2: Anti-alpha Tubulin mouse MAb (ab7291) at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution.

  • Anti-Histone H3 (tri methyl K27) antibody [EPR18607] - ChIP Grade (ab192985) at 1/1000 dilution + NIH/3T3 (Mouse embryonic fibroblast cell line) whole cell lysate at 10 µg

    Secondary
    Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/50000 dilution

    Predicted band size : 15 kDa
    Observed band size : 15 kDa


    Exposure time : 1 second

    Blocking/Dilution buffer: 5% NFDM/TBST.

  • Anti-Histone H3 (tri methyl K27) antibody [EPR18607] - ChIP Grade (ab192985) at 1/1000 dilution + HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysate at 10 µg

    Secondary
    Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/50000 dilution

    Predicted band size : 15 kDa
    Observed band size : 15 kDa


    Exposure time : 3 minutes

    Blocking/Dilution buffer: 5% NFDM/TBST.

  • All lanes : Anti-Histone H3 (tri methyl K27) antibody [EPR18607] - ChIP Grade (ab192985) at 1/1000 dilution

    Lane 1 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate
    Lane 2 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate with H3K4Me1 peptide at 5 µg
    Lane 3 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate with H3K4Me2 peptide at 5 µg
    Lane 4 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate with H3K4Me3 peptide at 5 µg
    Lane 5 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate with H3K9Me1 peptide at 5 µg
    Lane 6 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate with H3K9Me2 peptide at 5 µg
    Lane 7 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate with H3K9Me3 peptide at 5 µg
    Lane 8 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate with H3K27Me1 peptide at 5 µg
    Lane 9 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate with H3K27Me2 peptide at 5 µg
    Lane 10 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate with H3K27Me3 peptide at 5 µg
    Lane 11 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate with H3K27 unmodified peptide at 5 µg

    Lysates/proteins at 10 µg per lane.

    Secondary
    Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution
    Developed using the ECL technique

    Predicted band size : 15 kDa


    Exposure time : 5 seconds

    The antibody is blocked by tri methyl K27 peptide (lane 10) and slightly by di methyl K27 peptide (lane 9, there is 14% cross reactivity with di methyl K27 as determined by Elisa).

  • All lanes : Anti-Histone H3 (tri methyl K27) antibody [EPR18607] - ChIP Grade (ab192985) at 1/1000 dilution

    Lane 1 : HeLa (Human epithelial cell line from cervix adenocarcinoma)
    Lane 2 : EED-/- mouse whole cell lysate
    Lane 3 : Wild type mouse ES whole cell lysate

    Lysates/proteins at 10 µg per lane.

    Secondary
    Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/50000 dilution
    Developed using the ECL technique

    Predicted band size : 15 kDa


    Exposure time : 8 minutes
  • Peptide array analysis was performed using ab192985 at a concentration of 0.1ug/ml, followed by Goat Anti-Rabbit IgG, (H+L), Fluo 647nm conjugated secondary antibody at a 1/50000 dilution.

    All batches of ab192985 are tested in Peptide Array against 501 different modified and unmodified histone peptides; each peptide is printed on the array at six concentrations (each in triplicate).

    Circle area represents affinity between the antibody and a peptide: all antigen-containing peptides are displayed as red circles, all other peptides as blue circles. The affinity is calculated as area under curve when antibody binding values are plotted against the corresponding peptide concentration. Each circle area is normalized to the peptide with the strongest affinity.

    The complete dataset, including full list of all peptides and information on the position of each peptide in the diagram, can be downloaded here.

  • ELISA analysis was performed on 1ug/ml of antigen using ab192985 at a concentration range of 0-0.25ug/ml, followed by Alkaline Phosphatase-conjugates AffiniPure Goat anti-rabbit IgI (H&L) secondary antibody at a 1/2500 dilution.

    All batches of ab192985 are tested in ELISA against peptides to different Histone H3 modifications. Results show strong binding to Histone H3 - tri methyl K27 immunizing peptide, indicating that this antibody specifically recognizes the Histone H3 - tri methyl K27 modification. Weak binding (14%) was also detected against H3 - di methyl K27 modification.

  • Immunohistochemical analysis of paraffin-embedded human colon tissue labeling Histone H3 (tri methyl K27) with ab192985 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Nuclear staining on human colon tissue is observed. Counter stained with Hematoxylin.

    Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

  • Immunohistochemical analysis of paraffin-embedded mouse colon tissue labeling Histone H3 (tri methyl K27) with ab192985 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Nuclear staining on mouse colon tissue is observed. Counter stained with Hematoxylin.

    Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

  • Immunohistochemical analysis of paraffin-embedded rat kidney tissue labeling Histone H3 (tri methyl K27) with ab192985 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Nuclear staining on rat kidney tissue is observed. Counter stained with Hematoxylin.

    Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

References

This product has been referenced in:
  • Yang A  et al. Folate Protects Hepatocytes of Hyperhomocysteinemia Mice from Apoptosis via Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)-activated Endoplasmic Reticulum Stress. J Cell Biochem N/A:N/A (2017). ChIP ; Mouse . Read more (PubMed: 28230279) »

See 1 Publication for this product

Customer reviews and Q&As

Application
Flow Cytometry
Sample
Zebrafish Cell (Dissociated cells from zebrafish testicle)
Permeabilization
Yes - Methanol 90%
Gating Strategy
Cell populations were gated according to their DNA content
Specification
Dissociated cells from zebrafish testicle
Preparation
Cell harvesting/tissue preparation method: Cells were disgregated with collagenase and after filtration and fixation were subjected to immunocytochemistry
Sample buffer: Final buffer PBS
Fixation
Paraformaldehyde
Username

Abcam user community

Verified customer

Submitted Aug 01 2017

Application
Immunohistochemistry (Frozen sections)
Sample
Mouse Tissue sections (kidney)
Permeabilization
Yes - 0.05% Triton X-100
Specification
kidney
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 21°C
Fixative
10% normal buffered formalin
Username

Abcam user community

Verified customer

Submitted Jun 27 2017

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Human Tissue sections (stomach cancer)
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: TE pH9
Permeabilization
No
Specification
stomach cancer
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 22°C
Fixative
10% normal buffered formalin
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Abcam user community

Verified customer

Submitted Jun 16 2017

Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Mouse Tissue sections (Tumor)
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: TE pH9
Permeabilization
No
Specification
Tumor
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 21°C
Fixative
10% normal buffered formalin
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Abcam user community

Verified customer

Submitted Jun 12 2017

Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Rat Tissue sections (Lung with tumor)
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: TE pH9
Permeabilization
No
Specification
Lung with tumor
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 21°C
Fixative
10% normal buffered formalin
Username

Abcam user community

Verified customer

Submitted Jun 12 2017

Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Pig Tissue sections (Lung)
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: TE pH9
Permeabilization
No
Specification
Lung
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 21°C
Fixative
10% normal buffered formalin
Username

Abcam user community

Verified customer

Submitted Jun 12 2017

Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"

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