Overview

  • Product name
    Anti-Histone H3 (tri methyl K36) antibody - ChIP Grade
    See all Histone H3 primary antibodies
  • Description
    Rabbit polyclonal to Histone H3 (tri methyl K36) - ChIP Grade
  • Host species
    Rabbit
  • Specificity
    Histone H3 tri methylated at lysine 36
  • Tested applications
    Suitable for: ICC/IF, CHIPseq, WB, ChIP, ChIP/Chip, IHC-Pmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Cow, Human, Saccharomyces cerevisiae, Xenopus laevis, Arabidopsis thaliana, Caenorhabditis elegans, Drosophila melanogaster, Schizosaccharomyces pombe, Zebrafish, Silk worm, Rice, Xenopus tropicalis, Trypanosoma brucei
    Predicted to work with: Plants
  • Immunogen

    Synthetic peptide within Human Histone H3 aa 1-100 (tri methyl K36) conjugated to Keyhole Limpet Haemocyanin (KLH). The exact sequence is proprietary.
    (Peptide available as ab1785)

  • Positive control
    • WB: Calf thymus histone preparation and HeLa whole cell extract. ICC/IF: HeLa cells.
  • General notes

    For detection of methylated histone H3.

Properties

Applications

Our Abpromise guarantee covers the use of ab9050 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
ICC/IF Use a concentration of 0.1 - 1 µg/ml.

We recommend using Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed (ab150081) secondary antibody.

CHIPseq Use at an assay dependent concentration. PubMed: 19581485
WB Use a concentration of 1 µg/ml. Detects a band of approximately 15 kDa (predicted molecular weight: 15 kDa).
ChIP Use 4µg for 106 cells.
ChIP/Chip Use at an assay dependent concentration.
IHC-P Use a concentration of 0.5 - 10 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
  • Alternative names
    • 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

Images

  • ChIP analysis of H3K36 trimethylation (me3) along ctt1 in exo2Δ cells.

    Strains YAM2400 (WT) and YAM2402 (exo2Δ) were grown to mid-log phase in rich medium and collected before or after addition of 1 mM H2O2 for 15 minutes ctt1 mRNA and U3B snoRNA were detected from total RNA using 32P-labelled oligonucleotides. Data analysis was performed for each position, data were first normalized on act1, then on the level of histone H3, immunoprecipitated from the same chromatin. 

    Average values and SEM were calculated from three biological replicates.

    *p<0.05; ns, not significant upon t-test.

  • RNA Pol II phosphoepitopes and histone H3 modifications associated with transcription elongation are altered by sig-7(RNAi).

    Equal amounts of lysates from L4440 control RNAi and sig-7(RNAi) embryos were analyzed by western blots probed with ab9050 and antibodies against H3K79me2, and the 5’ end of active genes, H3K4me3. Antibodies against total histone H3 (H3) show similar protein loading.

  • Chromatin was prepared from U-2 OS (Human bone osteosarcoma epithelial cell line) cells according to the Abcam X-ChIP protocol.

    Cells were fixed with formaldehyde for 10 minutes. The  ChIP was performed with 25 µg of chromatin, 2 µg of ab9050 (blue), and 20 µl of Protein A/G sepharose beads. No antibody was added to the beads control (yellow). The immunoprecipitated DNA was quantified on the GAPDH (active) and MYO-D (inactive) promoters and over the ý-Actin gene (active).

    Schematic diagram of the ý-Actin gene is shown on the top of the figure.

    Black boxes represent exons and thin lines represent introns.

    PCR products are depicted as bars under the gene.

  • ChIP using ab9050 at the Pho4 locus in S. pombe.

    Chromatin extract from S.pombe cells was incubated with 5 µg of ab9050 overnight, and then incubated with Protein A beads for 1 hour. The immunoprecipitated DNA was quantified at the Pho4 locus by RT-PCR. No antibody was added to the beads control (red bars). A schematic diagram of the pho4 gene is shown below the graph. The grey box represents the open reading frame and PCR products are depicted by horizontal arrows.

    Please see anonymous abreview submitted on 29 October 2008 for additional details.

    See Abreview

  • ab9050 stained in HeLa (Human epithelial cell line from cervix adenocarcinoma) cells.

    Cells were fixed with 100% methanol for 5 minutes at room temperature and incubated with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% triton for 1 hour at room temperature to permeabilize the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody ab9050 at 0.1 µg/ml and ab7291 (Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control) at 1/1000 dilution overnight at +4°C. The secondary antibodies were Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed (ab150120) (pseudo-colored red) and Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed (ab150081) secondary antibody (colored green) used at 1 µg/ml for 1 hour at room temperature.

    DAPI was used to stain the cell nuclei (colored blue) at a concentration of 1.43 µM for 1 hour at room temperature.

  • All lanes : Anti-Histone H3 (tri methyl K36) antibody - ChIP Grade (ab9050) at 1 µg/ml

    Lane 1 : Calf Thymus Histone Preparation Nuclear Lysate
    Lane 2 : Calf Thymus Histone Preparation Nuclear Lysate with Histone H3 peptide - unmodified K36 at 0.5 µg/ml
    Lane 3 : Calf Thymus Histone Preparation Nuclear Lysate with Human Histone H3 (mono methyl K36) peptide (ab1783) at 0.5 µg/ml
    Lane 4 : Calf Thymus Histone Preparation Nuclear Lysate with Human Histone H3 (di methyl K36) peptide (ab1784) at 0.5 µg/ml
    Lane 5 : Calf Thymus Histone Preparation Nuclear Lysate with Human Histone H3 (tri methyl K36) peptide (ab1785) at 0.5 µg/ml
    Lane 6 : Calf Thymus Histone Preparation Nuclear Lysate with Human Histone H3 (tri methyl K37) peptide (ab24417) at 0.5 µg/ml

    Lysates/proteins at 0.5 µg per lane.

    Secondary
    All lanes : Goat polyclonal to Rabbit 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?

  • ab9050 staining Histone H3 (tri-methyl K36) in HeLa (Human epithelial cell line from cervix adenocarcinoma) cells.

    The cells were fixed with 100% methanol for 5 minutes and then blocked in 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1 hour. The cells were then incubated with ab9050 at 0.1 µg/ml and ab7291 (anti beta-Tubulin) at 1 µg/ml overnight at +4°C, followed by a further incubation at room temperature for 1 hour with a goat anti-rabbit AlexaFluor®488 secondary (ab150081) at 2 μg/ml (shown in green) and a goat anti-mouse AlexaFluor®594 (ab150120) at 2 μg/ml (shown in pseudo color red). Nuclear DNA was labeled in blue with DAPI.

    Negative controls: 1– Rabbit primary antibody and anti-mouse secondary antibody; 2 – Mouse primary antibody and anti-rabbit secondary antibody. Controls 1 and 2 indicate that there is no unspecific reaction between primary and secondary antibodies used.

  • ab9050 staining MAFA 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) for 3 hours at 25°C. An Alexa Fluor®555  conjugated Donkey anti-rabbit IgG polyclonal (1/250) was used as the secondary antibody.

    See Abreview

  • ab9050 staining rat liver tissue sections by IHC-P.

    The section was fixed in formaldehyde and subjected to heat mediated antigen retrieval in pH 6.0 citrate buffer prior to being blocked with 5% serum for 30 minutes at 45°C.  The primary antibody was diluted 1/500 and incubated for 45 minutes at 20°C.  An HRP conjugated goat anti-rabbit was used as the secondary

    See Abreview

  • ab9050 staining Histone H3 (tri methyl K36) in Saos-2 (Human osteosarcoma cell line) cells by ICC/IF (Immunocytochemistry/immunofluorescence).

    Cells were fixed with paraformaldehyde, permeabilized with 0.25% Triton in PBS and blocked with 1% BSA for 1 hour at room temperature. Samples were incubated with primary antibody (1/1000) for 1 hour. An Alexa Fluor®488-conjugated Goat anti-rabbit IgG polyclonal (1/250) was used as the secondary antibody.

    See Abreview

References

This product has been referenced in:
See all 562 Publications for this product

Customer reviews and Q&As

1-10 of 64 Abreviews or Q&A

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Human Cell lysate - whole cell (HeLa)
Gel Running Conditions
Non-reduced Denaturing (18%)
Loading amount
5 µg
Specification
HeLa
Blocking step
BSA as blocking agent for 12 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 4°C

Abcam user community

Verified customer

Submitted Dec 19 2017

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (HT1080)
Permeabilization
Yes - 0.1% Triton X100
Specification
HT1080
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 20°C
Fixative
Formaldehyde

Abcam user community

Verified customer

Submitted Nov 30 2017

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
ChIP
Sample
Human Cell lysate - nuclear (HT1080)
Negative control
IgG control
Specification
HT1080
Detection step
Real-time PCR
Type
Cross-linking (X-ChIP)
Duration of cross-linking step: 15 minute(s) and 0 second(s)
Specification of the cross-linking agent: PFA
Positive control
IP H3K36 ab

Abcam user community

Verified customer

Submitted Nov 30 2017

Application
ChIP
Sample
Human Cell lysate - nuclear (B cell lymphoma)
Negative control
IgG
Specification
B cell lymphoma
Detection step
Real-time PCR
Type
Cross-linking (X-ChIP)
Duration of cross-linking step: 5 minute(s) and 0 second(s)
Specification of the cross-linking agent: Methanol free formaldehyde
Positive control
Gene-Specific primets

Abcam user community

Verified customer

Submitted Nov 13 2017

Application
Immunoprecipitation
Sample
Human Cell lysate - nuclear (Human Embryonic Kidney (HEK293T))
Total protein in input
500 µg
Immuno-precipitation step
Other - Protein G Magnetic Beads
Specification
Human Embryonic Kidney (HEK293T)

Abcam user community

Verified customer

Submitted Sep 14 2017

Application
ChIP
Sample
Human Cell lysate - other (Epithelial cells)
Negative control
IgG
Specification
Epithelial cells
Detection step
Real-time PCR
Type
Cross-linking (X-ChIP)
Duration of cross-linking step: 10 minute(s) and 0 second(s)
Positive control
Input DNA

Abcam user community

Verified customer

Submitted Sep 30 2015

Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Mouse Tissue sections (Liver)
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: Citrate buffer ph6 20 min at 97ºC in a PT link
Permeabilization
No
Specification
Liver
Blocking step
Serum as blocking agent for 30 minute(s) · Concentration: 10% · Temperature: 25°C
Fixative
Formaldehyde

Miss. Virginia Gutiérrez

Verified customer

Submitted Sep 24 2015

Application
ChIP
Sample
Mouse Cell lysate - nuclear (mES E14)
Negative control
IgG ChIP
Specification
mES E14
Detection step
Real-time PCR
Type
Cross-linking (X-ChIP)
Duration of cross-linking step: 10 minute(s) and 0 second(s)
Specification of the cross-linking agent: PAF

Abcam user community

Verified customer

Submitted Aug 12 2015

Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: Na-citrate pH6
Sample
Mouse Tissue sections (Liver, P5)
Specification
Liver, P5
Permeabilization
Yes - Triton 0,05%
Fixative
Paraformaldehyde

Abcam user community

Verified customer

Submitted Oct 07 2014

Application
Immunocytochemistry/ Immunofluorescence
Blocking step
Serum as blocking agent for 10 minute(s) · Concentration: 1% · Temperature: 21°C
Sample
Human Cell (HEK293)
Specification
HEK293
Permeabilization
Yes - triton
Fixative
Paraformaldehyde

Abcam user community

Verified customer

Submitted Apr 25 2014

1-10 of 64 Abreviews or Q&A

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