Anti-Histone H3 (mono methyl K9) antibody - ChIP Grade (ab8896)

Overview

  • Product nameAnti-Histone H3 (mono methyl K9) antibody - ChIP GradeSee all Histone H3 primary antibodies ...
  • Description
    Rabbit polyclonal to Histone H3 (mono methyl K9) - ChIP Grade
  • SpecificitySpecific for mono-methyl lysine 9 of Histone H3. There is no cross-reactivity with lysine 27 of Histone H3.
  • Tested applicationsWB, ChIP, ICC/IF, CHIPseq, Flow Cytmore details
  • Species reactivity
    Reacts with: Cow, Human, Caenorhabditis elegans
    Predicted to work with: a wide range of other species, all Mammals
  • Immunogen

    Synthetic peptide derived from within residues 1 - 100 Histone H3.

    (Peptide available as ab1771.)

  • Positive control
    • Calf Thymus Histone Preparation This antibody gave a positive result when used in the following methanol fixed cell lines: HeLa.

Properties

  • FormLiquid
  • Storage instructionsShipped 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.
  • Storage bufferpH: 7.40
    Preservative: 0.02% Sodium azide
    Constituent: PBS
    Note: 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...
  • PurityImmunogen affinity purified
  • Clonality Polyclonal
  • IsotypeIgG
  • Research Areas

Applications

Our Abpromise guarantee covers the use of ab8896 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
WB Use a concentration of 0.5 - 2 µg/ml. Detects a band of approximately 17 kDa (predicted molecular weight: 15 kDa).Can be blocked with Human Histone H3 (mono methyl K9) peptide (ab1771).
ChIP Use 2-25 µg for µg of chromatin.
ICC/IF Use at an assay dependent concentration.
CHIPseq Use at an assay dependent concentration. PubMed: 22196736
Flow Cyt Use at an assay dependent concentration.

Target

  • FunctionCore 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 similaritiesBelongs to the histone H3 family.
  • Developmental stageExpressed 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 localizationNucleus. Chromosome.
  • Information by UniProt
  • Database links
  • Alternative names
    • H3 histone family, member J antibody
    • FLJ92264 antibody
    • H3 histone family member E pseudogene antibody
    • H3 histone family member E pseudogene 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 3A 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
    • H31_HUMAN antibody
    • H3F1K antibody
    • H3F3 antibody
    • H3F3 antibody
    • H3FA antibody
    • H3FB antibody
    • H3FC antibody
    • H3FD antibody
    • H3FF antibody
    • H3FH antibody
    • H3FI antibody
    • H3FJ antibody
    • H3FK antibody
    • H3FL antibody
    • HIST1H3A antibody
    • HIST1H3B antibody
    • HIST1H3C antibody
    • HIST1H3D antibody
    • HIST1H3E antibody
    • HIST1H3F antibody
    • HIST1H3G antibody
    • HIST1H3H antibody
    • HIST1H3I antibody
    • HIST1H3J antibody
    • HIST3H3 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 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 H 3 antibody
    • Histone H3 3 pseudogene antibody
    • Histone H3 3 pseudogene antibody
    • Histone H3.1 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

Anti-Histone H3 (mono methyl K9) antibody - ChIP Grade images

  • All lanes : Anti-Histone H3 (mono methyl K9) antibody - ChIP Grade (ab8896) at 1 µg/ml

    Lane 1 : Calf Thymus Histone Preparation
    Lane 2 : Calf Thymus Histone Preparation with Human Histone H3 (mono methyl K9) peptide (ab1771) at 0.5 µg/ml
    Lane 3 : Calf Thymus Histone Preparation with Human Histone H3 (di methyl K9) peptide (ab1772) at 0.5 µg/ml
    Lane 4 : Calf Thymus Histone Preparation with Human Histone H3 (tri methyl K9) peptide (ab1773) at 0.5 µg/ml
    Lane 5 : Calf Thymus Histone Preparation with Histone H3 (mono methyl K27) peptide (ab1780) at 0.5 µg/ml
    Lane 6 : Calf Thymus Histone Preparation with Human Histone H3 (di methyl K27) peptide (ab1781) at 0.5 µg/ml
    Lane 7 : Calf Thymus Histone Preparation with Human Histone H3 (tri methyl K27) peptide (ab1782) at 0.5 µg/ml
    Lane 8 : Calf Thymus Histone Preparation with Human Histone H3 (mono methyl K4) peptide (ab1340) at 0.5 µg/ml
    Lane 9 : Calf Thymus Histone Preparation with Human Histone H3 (unmodified ) peptide (ab7228) at 0.5 µg/ml
    Lane 10 : Calf Thymus Histone Preparation with Human Histone H3 (unmodified ) peptide (ab2623) at 0.5 µg/ml

    Lysates/proteins at 0.5 µg per lane.

    Secondary
    Goat polyclonal to Rabbit IgG H&L (HRP) Pre-Adsorbed at 1/10000 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?)

    ab8896 specifically recognises mono-methyl K9 (lane 1) in calf thymus lysate at 17kDa. ab8896 is successfully blocked using the immunizing peptide (lane 2 ab1771), but not the di-methyl K9 (lane 3 ab1772), the tri-methyl K9 (lane 4 ab1773), the mono-methyl K27 (lane 5 ab1780), the di-methyl K27 (lane 6 ab1781), the tri-methyl K27 (lane 7 ab1782), the mono-methyl K4 (lane 8 ab1340) nor the corresponding unmodified peptides (lane 9 ab7228, lane 10 ab2623). This implies that ab8896 is specific to mono-methyl K9.

    See Abreview

  • 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, 2µg of  ab8896 (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 for active and inactive loci, Sybr green approach for heterochromatic loci). Primers and probes are located in the first kb of the transcribed region.

  • ab8896 staining Histone H3 (mono methyl K9) in human differentiated haematopoietic stem cells by Flow Cytometry. Cells were fixed with paraformaldehyde and permeabilized with permeabilization buffer. The sample was incubated with the primary antibody (1/600) for 12 hours at 4°C. An Alexa Fluor® 488-conjugated goat polyclonal anti-rabbit IgG (1/1000) was used as the secondary antibody.
    Gating Strategy: Isotype negative control (white).

    See Abreview

  • ICC/IF image of ab8896 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 ab8896 at 1µg/ml overnight at +4°C. The secondary antibody (pseudo-colored green) was Alexa Fluor® 488 goat anti- rabbit (ab150081) IgG (H+L) preadsorbed, used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (pseudo-colored red) at a 1/200 dilution for 1h at room temperature. DAPI was used to stain the cell nuclei (pseudo-colored blue) at a concentration of 1.43µM for 1hour at room temperature.

  • All lanes : Anti-Histone H3 (mono methyl K9) antibody - ChIP Grade (ab8896) at 1/1000 dilution

    Lane 1 : Nuclear lysates prepared from Hela cell: SiRNA untreated
    Lane 2 : Nuclear lysates prepared from Hela cells treated with G9a SiRNA

    Lysates/proteins at 10 µg per lane.

    Secondary
    Alkaline Phosphatase conjugated rabbit polyclonal rabbit IgG (Fc) at 1/5000 dilution

    Predicted band size : 15 kDa
    Observed band size : 16 kDa (why is the actual band size different from the predicted?)


    Exposure time : 10 minutes

    The image is a courtesy of an anonymous abreview.

References for Anti-Histone H3 (mono methyl K9) antibody - ChIP Grade (ab8896)

This product has been referenced in:
  • Baas R  et al. A novel microscopy-based high-throughput screening method to identify proteins that regulate global histone modification levels. J Biomol Screen 19:287-96 (2014). WB, ICC/IF ; Human . Read more (PubMed: 24334265) »
  • Wang L  et al. Histone H3K9 methyltransferase G9a represses PPAR? expression and adipogenesis. EMBO J 32:45-59 (2013). Read more (PubMed: 23178591) »

See all 14 Publications for this product

Product Wall

Application Flow Cytometry
Fixation Paraformaldehyde
Permeabilization Yes - eBioscience Permeabilization Buffer
Sample Human Cell (Differentiated Haematopoietic Stem Cells)
Specification Differentiated Haematopoietic Stem Cells
Gating Strategy Isotype negative control (white)
Preparation Cell harvesting/tissue preparation method: Typsin-EDTA Cell Dissociation
Sample buffer: PBS and 10% FBS
Username

Abcam user community

Verified customer

Submitted Feb 20 2014

Application Western blot
Loading amount 20 µg
Gel Running Conditions Reduced Denaturing
Sample Human Tissue lysate - whole (Patient Dermal Fibroblasts)
Specification Patient Dermal Fibroblasts
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 22°C
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Submitted Feb 05 2014

Application Western blot
Loading amount 4.5 µg
Gel Running Conditions Reduced Denaturing (12)
Sample Caenorhabditis elegans Tissue lysate - nuclear (C. elegans whole body)
Specification C. elegans whole body
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 23°C
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Submitted Oct 28 2013

Application Western blot
Loading amount 20 µg
Gel Running Conditions Reduced Denaturing (12)
Sample Human Cell lysate - whole cell (Human HeLa cells)
Specification Human HeLa cells
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 23°C
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Submitted Oct 28 2013

Application Immunocytochemistry/ Immunofluorescence
Sample Human Cell (HeLa)
Specification HeLa
Fixative Paraformaldehyde
Permeabilization Yes - 0.05% Triton X100 in PBS
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Dr. Kirk McManus

Verified customer

Submitted Feb 19 2013

Thank you for taking time to complete our questionnaire and for contacting us. I am sorry to hear this antibody is not providing satisfactory results.

The details provided will enable us to investigate this case and will provide us with vi...

Read More
Application Western blot
Sample Human Cell lysate - whole cell (huh-7 cells)
Loading amount 15 µg
Specification huh-7 cells
Treatment cells expressing recombitant protein
Gel Running Conditions Reduced Denaturing
Blocking step Milk as blocking agent for 1 minute(s) · Concentration: 0.5% · Temperature: 20°C
Username

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Submitted Jan 04 2012

Application Immunocytochemistry/ Immunofluorescence
Sample Human Cell (HeLa (cervical cancer cell line))
Specification HeLa (cervical cancer cell line)
Fixative Paraformaldehyde
Permeabilization Yes - 0.3% Triton/TBS
Blocking step Serum as blocking agent for 30 minute(s) · Concentration: 2% · Temperature: 25°C
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Submitted Dec 15 2010

Application ChIP
Sample Human Cell lysate - nuclear (K562)
Specification K562
Type Cross-linking (X-ChIP)
Duration of cross-linking step: 10 minute(s) and 0 second(s)
Specification of the cross-linking agent: 1% formaldehyde
Detection step Semiquantitative PCR
Positive control USP3
Negative control HNRPA2B1 was used as a negative control as seen using publicly available ChIP-Seq data in K562 cells.
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Submitted Nov 01 2010

Application ChIP
Sample Human Cell lysate - nuclear (HeLa)
Specification HeLa
Type Cross-linking (X-ChIP)
Duration of cross-linking step: 10 minute(s) and 0 second(s)
Specification of the cross-linking agent: 1% formaldehyde
Detection step Real-time PCR
Positive control bglobin
Negative control GAPDH
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Submitted Mar 18 2010

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