Anti-Histone H3 (di methyl K4, tri methyl K4) antibody [mAbcam 6000] - ChIP Grade (ab6000)

Reviews (7)Q&A (1)References (28)

Overview

  • Product name

    Anti-Histone H3 (di methyl K4, tri methyl K4) antibody [mAbcam 6000] - ChIP Grade
    See all Histone H3 primary antibodies

  • Description

    Mouse monoclonal [mAbcam 6000] to Histone H3 (di methyl K4, tri methyl K4) - ChIP Grade

  • Host species

    Mouse

  • Specificity

    This antibody detects a band of the appropriate size when used in Western blotting on a calf thymus histone preparation (see image). It is specifically blocked by peptides corresponding to di methylated K4 of Histone H3 and to tri methylated K4. Slight cross reactivity is observed with mono methyl K4 peptide in ELISA and Western Blot.

  • Tested applications

    Suitable for: ICC/IF, IHC-P, WB, ICC, ChIP, Flow Cytmore details

  • Species reactivity

    Reacts with: Mouse, Rabbit, Cow, Human, Saccharomyces cerevisiae, Drosophila melanogaster, Rice, Candida albicans
    Predicted to work with: Rat, Chicken, Xenopus laevis, Arabidopsis thaliana, Caenorhabditis elegans, Schizosaccharomyces pombe, Neurospora crassa

  • Immunogen

    Synthetic peptide within Human Histone H3 aa 1-100 (tri methyl K4) conjugated to keyhole limpet haemocyanin (Sulfosuccinimidyl 4-N-maleimidomethyl-cyclohexane-1-carboxylate (Sulfo-SMCC)). The exact sequence is proprietary. Hybridomas were prepared and the resulting clones were positively screened by ELISA against the immunising peptide and negatively screened against the non-modified equivalent peptide.
    (Peptide available as ab1342, ab7768)

  • Positive control

    • ChIP: Chromatin was prepared from U-2 OS cell . FC: HeLa cells. WB: Calf thymus histone preparation nuclear lysate.

  • General notes

    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.

Properties

Applications

Our Abpromise guarantee covers the use of ab6000 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 1 µg/ml.
IHC-P Use at an assay dependent concentration. PubMed: 20823885
WB 1/500. Predicted molecular weight: 15.2 kDa.Can be blocked with Human Histone H3 (tri methyl K4) peptide (ab1342) or Human Histone H3 (di methyl K4) peptide (ab7768).
ICC 1/200.
ChIP Use at an assay dependent concentration.
Flow Cyt Use 1µg for 106 cells.

ab170192 - Mouse monoclonal IgG2b, is suitable for use as an isotype control with this antibody.

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

    • 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 - Anti-Histone H3 (di methyl K4, tri methyl K4) antibody [mAbcam 6000] - ChIP Grade (ab6000)
    ChIP - Anti-Histone H3 (di methyl K4, tri methyl K4) antibody [mAbcam 6000] - ChIP Grade (ab6000)

    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  ab6000 (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.

  • Immunocytochemistry/ Immunofluorescence - Anti-Histone H3 (di methyl K4, tri methyl K4) antibody [mAbcam 6000] - ChIP Grade (ab6000)
    Immunocytochemistry/ Immunofluorescence - Anti-Histone H3 (di methyl K4, tri methyl K4) antibody [mAbcam 6000] - ChIP Grade (ab6000)This image is courtesy of Luke Hughes-Davies and Rhiannon Jade, Gurdon Institute, Cambridge, UK

    Immunofluorescent imaging of human cells (U2OS) with ab6000 reveals a diffuse background nuclear staining corresponding to global dimethylation of K4, with multiple foci of brighter staining.  The complete lack of nucleolar or cytoplasmic staining background confirms the high specificity of this antibody in this application.   

    IF was performed with a standard paraformaldehyde technique (fixed in PBS buffered PFH 4% for 5 minutes, permeabilised with 0.5% triton-PBS for 5 minutes, blocked with 5% milk / 0.2% tween for one hour.  Primary antibody used at 1/200 in 5% milk / 0.2% TWEEN for one hour, secondary antibody Alexa 488 for 30 minutes.  All blocking and incubation steps carried out at 37 degrees.

    Due to the size constraints for images on our website, unfortunately we are unable to show a higher quality image.

  • Western blot - Anti-Histone H3 (di methyl K4, tri methyl K4) antibody [mAbcam 6000] - ChIP Grade (ab6000)
    Western blot - Anti-Histone H3 (di methyl K4, tri methyl K4) antibody [mAbcam 6000] - ChIP Grade (ab6000)
    All lanes : Anti-Histone H3 (di methyl K4, tri methyl K4) antibody [mAbcam 6000] - ChIP Grade (ab6000) at 1 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 0.5 µg
    Lane 3 : Calf Thymus Histone Preparation Nuclear Lysate with Human Histone H3 (mono methyl K4) peptide (ab1340) at 0.5 µg
    Lane 4 : Calf Thymus Histone Preparation Nuclear Lysate with Human Histone H3 (di methyl K4) peptide (ab7768) at 0.5 µg
    Lane 5 : Calf Thymus Histone Preparation Nuclear Lysate with Human Histone H3 (tri methyl K4) peptide (ab1342) at 0.5 µg
    Lane 6 : Calf Thymus Histone Preparation Nuclear Lysate with Human Histone H3 (mono methyl K9) peptide (ab1771) at 0.5 µg
    Lane 7 : Calf Thymus Histone Preparation Nuclear Lysate with Human Histone H3 (di methyl K9) peptide (ab1772) at 0.5 µg
    Lane 8 : Calf Thymus Histone Preparation Nuclear Lysate with Human Histone H3 (tri methyl K9) peptide (ab1773) at 0.5 µg
    Lane 9 : Calf Thymus Histone Preparation Nuclear Lysate with Human Histone H3 (mono methyl K27) peptide (ab1780) at 0.5 µg
    Lane 10 : Calf Thymus Histone Preparation Nuclear Lysate with Human Histone H3 (di methyl K27) peptide (ab1781) at 0.5 µg
    Lane 11 : Calf Thymus Histone Preparation Nuclear Lysate with Human Histone H3 (tri methyl K27) peptide (ab1782) at 0.5 µg

    Lysates/proteins at 0.5 µg per lane.

    Secondary
    All lanes : Goat Anti-Mouse IgG H&L (HRP) preadsorbed (ab97040) at 1/10000 dilution

    Developed using the ECL technique.

    Performed under reducing conditions.

    Predicted band size: 15.2 kDa
    Observed band size: 17 kDa
    why is the actual band size different from the predicted?


    Exposure time: 20 minutes
  • Flow Cytometry - Anti-Histone H3 (di methyl K4, tri methyl K4) antibody [mAbcam 6000] - ChIP Grade (ab6000)
    Flow Cytometry - Anti-Histone H3 (di methyl K4, tri methyl K4) antibody [mAbcam 6000] - ChIP Grade (ab6000)

    Overlay histogram showing HeLa cells stained with ab6000 (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 (ab6000, 1µg/1x106 cells) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG2b [PLPV219] (ab91366, 2µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in HeLa cells fixed with 4% paraformaldehyde (10 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

  • Immunocytochemistry/ Immunofluorescence - Anti-Histone H3 (di methyl K4, tri methyl K4) antibody [mAbcam 6000] - ChIP Grade (ab6000)
    Immunocytochemistry/ Immunofluorescence - Anti-Histone H3 (di methyl K4, tri methyl K4) antibody [mAbcam 6000] - ChIP Grade (ab6000)

    Left image shows human Hep cell monolayer fixed with 4% formaldehyde, permeabilized using 0.5% Triton X-100, blocked with 5% FCS in PBS (20 min) and incubated with ab6000 for 1 hr (diluted 1/200 in PBS plus 1% FCS). Detection with anti-mouse rhodamin labeled secondary antibody. Unstained areas represent nucleoli and heterochromatin as judged from DAPI overlay of the same cell in the right image.

    This image was kindly supplied as part of the review submitted by Christian Schoefer.

References

This product has been referenced in:

See all 28 Publications for this product

Publishing research using ab6000? Please let us know so that we can cite the reference in this datasheet.

Customer reviews and Q&As

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1-8 of 8 Abreviews or Q&A

Western blot abreview for Anti-Histone H3 (methylated) antibody [mAbcam 6000] - ChIP Grade

 Good
Abreviews
abreview image
Application
Western blot
Sample
Human Cell lysate - whole cell (Hela)
Loading amount
10 µg
Specification
Hela
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
Read More

Mr. Benny Garfinkel

Verified customer

Submitted Aug 05 2010

Immunocytochemistry/ Immunofluorescence abreview for Anti-Histone H3 (methylated) antibody [mAbcam 6000] - ChIP Grade

 Excellent
Abreviews
abreview image
Application
Immunocytochemistry/ Immunofluorescence
Sample
Mouse Cell (3T3 mouse fibroblasts)
Specification
3T3 mouse fibroblasts
Fixative
Paraformaldehyde
Permeabilization
Yes - 1% Triton X-100 4 minutes
Read More

Mr. Benny Garfinkel

Verified customer

Submitted Jul 16 2010

Immunocytochemistry/ Immunofluorescence abreview for Anti-Histone H3 (methylated) antibody [mAbcam 6000] - ChIP Grade

 Excellent
Abreviews
abreview image
Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (Hela)
Specification
Hela
Fixative
Paraformaldehyde
Read More

Mr. Benny Garfinkel

Verified customer

Submitted Jul 15 2010

ChIP abreview for Anti-Histone H3 (methylated) antibody [mAbcam 6000] - ChIP Grade

 Excellent
Abreviews
abreview image
Application
ChIP
Sample
Rice Cell lysate - whole cell (14 days old seedlings grown under dark condition)
Specification
14 days old seedlings grown under dark condition
Type
Native ChIP (N-ChIP)
Detection step
Real-time PCR
Positive control
Gene of interest
Negative control
No Antibody control
Read More

豊 佐藤

Verified customer

Submitted Dec 05 2007

Immunocytochemistry/ Immunofluorescence abreview for Anti-Histone H3 (methylated) antibody [mAbcam 6000] - ChIP Grade

 Good
Abreviews
abreview image
Application
Immunocytochemistry/ Immunofluorescence
Sample
Mouse Cell (MEFs SV40 transformed)
Specification
MEFs SV40 transformed
Fixative
Paraformaldehyde
Permeabilization
Yes - 0.5% triton 10 min RT
Blocking step
BSA as blocking agent for 30 minute(s) · Concentration: 1% · Temperature: RT°C
Read More

Mr. Mark Rochman

Verified customer

Submitted Aug 13 2007

Western blot abreview for Anti-Histone H3 (methylated) antibody [mAbcam 6000] - ChIP Grade

 Excellent
Abreviews
abreview image
Application
Western blot
Sample
Not applicable Cell lysate - whole cell (whole cell lysate - Candida albicans)
Loading amount
2 µg
Specification
whole cell lysate - Candida albicans
Blocking step
Milk as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 5%
Read More

Dr. Suresh Babu Raman

Verified customer

Submitted Mar 24 2006

Immunocytochemistry/ Immunofluorescence abreview for Anti-Histone H3 (methylated) antibody [mAbcam 6000] - ChIP Grade

 Inconclusive
Abreviews
Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (spermatocytes)
Specification
spermatocytes
Fixative
Paraformaldehyde
Blocking step
Other as blocking agent for 30 minute(s) · Concentration: 0.15%BSA+0
Read More

Abcam user community

Verified customer

Submitted Feb 27 2006

Question

I'm interesting to purchase antibodies against Histone 3 di methyl K4 (ab7766) or Histone H3 (di+tri methyl K4) ab6000, for the use in ChiP assay. The organism we are working is is Entamoeba histolytica and we know that the sequence around K4 is similar histones from other origins. But we do not know the degree of methylation (di or tri). Would you know any preference of using the di methyl which is polyclonal or the di+tri which is monoclonal for ChIP in order to distinguish between transcriptional active and silenced genes.

Read More
Answer

I have performed an alignment of Entamoeba histolytica and homo sapiens and indeed there does seem to be some similarity in the amino acid sequence at lysine 4 of H3 (ARTK). These antisera have yet to be tested in this species. However this is not to say that it will not work, just that it has yet to be tested. Entamoeba histolytica histone H3 (Q50XG0_ENTHI) Entamoeba MARTKGHIERPSNKSAKAVKNVAFKAA Homo sapi -ARTK-QTARKSTGGKAPRKQLATKAA ****.: * *. . . *::* *** ab7766 and ab6000 are £198 each and have both been tested using our own recommended ChIP protocol. This can be accessed at the following link: https://www.abcam.com/index.html?pageconfig=view_protocol&pid=171 Both antisera work well by ChIP. However, in your example I would recommend determining the presence of either modification using mass spec before embarking on ChIP experiments as negative results are difficult to prove given that these antisera have not been tested on your species of interest.

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