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)](https://www.abcam.com/ps/products/6/ab6000/Images/ab6000-11726-anti-histone-h3-di-methyl-k4-tri-methyl-k4-antibody-mabcam-6000-chip-grade-chip.jpg "ChIP - Anti-Histone H3 (di methyl K4, tri methyl K4) antibody [mAbcam 6000] - ChIP Grade (ab6000)")
Key features and details
- Mouse monoclonal [mAbcam 6000] to Histone H3 (di methyl K4, tri methyl K4) - ChIP Grade
- Suitable for: ICC/IF, WB, ChIP, Flow Cyt
- Reacts with: Cow, Human, Rice
- Isotype: IgG2b
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 & Species
See all applications and species dataApplication Species ChIP HumanFlow Cyt HumanICC/IF HumanWB Cow
-
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 asab1342, ab7768) -
Positive control
- ChIP: Chromatin was prepared from U-2 OS cell . Flow Cyt: 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.
The Life Science industry has been in the grips of a reproducibility crisis for a number of years. Abcam is leading the way in addressing the problem with our range of recombinant monoclonal antibodies and knockout edited cell lines for gold-standard validation.
One factor contributing to the crisis is the use of antibodies that are not suitable. This can lead to misleading results and the use of incorrect data informing project assumptions and direction. To help address this challenge, we have introduced an application and species grid on our primary antibody datasheets to make it easy to simplify identification of the right antibody for your needs.
Learn more here.
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 or -80°C. Avoid freeze / thaw cycle. -
Storage buffer
pH: 7.40
Preservative: 0.02% Sodium azide
Constituents: PBS, 6.97% L-Arginine -
Concentration information loading... -
Purity
IgG fraction -
Clonality
Monoclonal -
Clone number
mAbcam 6000 -
Myeloma
Sp2/0-Ag14 -
Isotype
IgG2b -
Light chain type
kappa -
Research areas
Associated products
-
Alternative Versions
-
ChIP Related Products
-
Compatible Secondaries
-
Corresponding Unmodified Peptide
-
Immunizing Peptide (Blocking)
-
Isotype control
-
Recombinant Protein
-
Related Products
Applications
The Abpromise guarantee
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.
Tested applications are guaranteed to work and covered by our Abpromise guarantee.
Predicted to work for this combination of applications and species but not guaranteed.
Does not work for this combination of applications and species.
| Application | Species |
|---|---|
| ChIP |
Human
|
| Flow Cyt |
Human
|
| ICC/IF |
Human
|
| WB |
Cow
|
| All applications |
Mouse
Rat
Rabbit
Chicken
Saccharomyces cerevisiae
Xenopus laevis
Arabidopsis thaliana
Caenorhabditis elegans
Drosophila melanogaster
Schizosaccharomyces pombe
Neurospora crassa
Candida albicans
|
| Application | Abreviews | Notes |
|---|---|---|
| ICC/IF | (4) |
Use a concentration of 1 µg/ml.
|
| WB | (2) |
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).
|
| ChIP | (1) |
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. |
| Notes |
|---|
|
ICC/IF
Use a concentration of 1 µg/ml. |
|
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). |
|
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
modificationsAcetylation 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
- Entrez Gene: 8350 Human
- Entrez Gene: 8351 Human
- Entrez Gene: 8352 Human
- Entrez Gene: 8353 Human
- Entrez Gene: 8354 Human
- Entrez Gene: 8355 Human
- Entrez Gene: 8356 Human
- Entrez Gene: 8357 Human
see all -
Alternative names
- H3 histone family member E pseudogene antibody
- H3 histone family, member A antibody
- H3/A antibody
see all
Images
-
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)](https://www.abcam.com/ps/products/6/ab6000/Images/ab6000-11727-anti-histone-h3-di-methyl-k4-tri-methyl-k4-antibody-mabcam-6000-chip-grade-immunofluorescence.jpg)
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, UKImmunofluorescent 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)](https://www.abcam.com/ps/products/6/ab6000/Images/ab6000-221321-anti-histone-h3-di-methyl-k4-tri-methyl-k4-antibody-mabcam-6000-chip-grade-western-blot.jpg)
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 withHuman Histone H3 (unmodified) peptide (ab7228) at 0.5 µg
Lane 3 : Calf Thymus Histone Preparation Nuclear Lysate withHuman Histone H3 (mono methyl K4) peptide (ab1340) at 0.5 µg
Lane 4 : Calf Thymus Histone Preparation Nuclear Lysate withHuman Histone H3 (di methyl K4) peptide (ab7768) at 0.5 µg
Lane 5 : Calf Thymus Histone Preparation Nuclear Lysate withHuman Histone H3 (tri methyl K4) peptide (ab1342) at 0.5 µg
Lane 6 : Calf Thymus Histone Preparation Nuclear Lysate withHuman Histone H3 (mono methyl K9) peptide (ab1771) at 0.5 µg
Lane 7 : Calf Thymus Histone Preparation Nuclear Lysate withHuman Histone H3 (di methyl K9) peptide (ab1772) at 0.5 µg
Lane 8 : Calf Thymus Histone Preparation Nuclear Lysate withHuman Histone H3 (tri methyl K9) peptide (ab1773) at 0.5 µg
Lane 9 : Calf Thymus Histone Preparation Nuclear Lysate withHuman Histone H3 (mono methyl K27) peptide (ab1780) at 0.5 µg
Lane 10 : Calf Thymus Histone Preparation Nuclear Lysate withHuman Histone H3 (di methyl K27) peptide (ab1781) at 0.5 µg
Lane 11 : Calf Thymus Histone Preparation Nuclear Lysate withHuman 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)](https://www.abcam.com/ps/products/6/ab6000/Images/ab6000-11724-anti-histone-h3-di-methyl-k4-tri-methyl-k4-antibody-mabcam-6000-chip-grade-flow-cytometry.jpg)
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)](https://www.abcam.com/ps/products/6/ab6000/Images/ab6000-11723-anti-histone-h3-di-methyl-k4-tri-methyl-k4-antibody-mabcam-6000-chip-grade-immunofluorescence.jpg)
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.
Datasheets and documents
References (31)
ab6000 has been referenced in 31 publications.
- Liu J et al. Dynamic patterns of H3K4me3, H3K27me3, and Nanog during rabbit embryo development. Am J Transl Res 11:430-441 (2019). PubMed: 30787999
- Kang Y et al. Effects of Salmonella enteritidis Vaccination on the Expression of Innate Immune Molecules and Histone Modifications in the Follicular Theca of Laying Hens. J Poult Sci 56:298-307 (2019). PubMed: 32055228
- Gibbons HR et al. Divergent lncRNA GATA3-AS1 Regulates GATA3 Transcription in T-Helper 2 Cells. Front Immunol 9:2512 (2018). PubMed: 30420860
- Wheaton K et al. UbE2E1/UBCH6 Is a Critical in Vivo E2 for the PRC1-catalyzed Ubiquitination of H2A at Lys-119. J Biol Chem 292:2893-2902 (2017). PubMed: 28073915
- Xiong Y et al. Effects of high progesterone level on the day of human chorionic gonadotrophin administration in in vitro fertilization cycles on epigenetic modification of endometrium in the peri-implantation period. Fertil Steril 108:269-276.e1 (2017). PubMed: 28778281
