Anti-Histone H3 (di methyl K4) antibody - ChIP Grade (ab11946)
- Datasheet
- References (16)
- Protocols
Overview
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Product name
Anti-Histone H3 (di methyl K4) antibody - ChIP Grade
See all Histone H3 primary antibodies -
Description
Goat polyclonal to Histone H3 (di methyl K4) - ChIP Grade -
Host species
Goat -
Tested applications
Suitable for: IHC-P, ChIP, ICC/IF, WBmore details -
Species reactivity
Reacts with: Mouse, Rat, Cow, Human, Saccharomyces cerevisiae, Arabidopsis thaliana, Caenorhabditis elegans
Predicted to work with: Chicken, Xenopus laevis, Drosophila melanogaster, Schizosaccharomyces pombe, Zebrafish, a wide range of other species, Tobacco, Chlamydomonas reinhardtii, Aspergillus nidulans, Neurospora crassa
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Immunogen
Synthetic peptide within Human Histone H3 aa 1-100 (di methyl K4) conjugated to keyhole limpet haemocyanin. The exact sequence is proprietary.
(Peptide available asab7768) -
Positive control
- This antibody gave a positive signal in Calf Thymus Histone Preparation as well as the following whole cell lysates: PC12; NIH3T3; MEF1. It also gave a positive signal in FFPE human normal cervix tissue sections
Properties
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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, 1% BSA
This product may contain up to 3% BSA depending on the batch. For specific batch formulations please contact us. -
Concentration information loading... -
Purity
Immunogen affinity purified -
Clonality
Polyclonal -
Isotype
IgG -
Research areas
Associated products
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ChIP Related Products
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Compatible Secondaries
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Immunizing Peptide (Blocking)
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Isotype control
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Recombinant Protein
Applications
Our Abpromise guarantee covers the use of ab11946 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 | Use a concentration of 1 µg/ml. Perform heat mediated antigen retrieval before commencing with IHC staining protocol. | |
| ChIP | Use 2-4 µg for 25 µg of chromatin. | |
| ICC/IF | 1/500. Use with paraformaldehyde or with methanol fixed cells. | |
| WB | Use a concentration of 1 µg/ml. Detects a band of approximately 17 kDa (predicted molecular weight: 15 kDa).Can be blocked with Human Histone H3 (di methyl K4) peptide (ab7768). |
Target
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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
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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
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ChIP - Anti-Histone H3 (di methyl K4) antibody - ChIP Grade (ab11946)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 ab11946 (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). Primers and probes are located in the first kb of the transcribed region.
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Western blot - Anti-Histone H3 (di methyl K4) antibody - ChIP Grade (ab11946)All lanes : Anti-Histone H3 (di methyl K4) antibody - ChIP Grade (ab11946) at 1 µg/ml
Lane 1 : NIH 3T3 (Mouse embryonic fibroblast cell line) Whole Cell Lysate
Lane 2 : MEF1 (Mouse embryonic fibroblast cell line) Whole Cell Lysate
Lysates/proteins at 15 µg per lane.
Secondary
All lanes : Rabbit polyclonal to Goat IgG - H&L - Pre-Adsorbed (HRP) (ab65486) at 1/3000 dilution
Developed using the ECL technique.
Performed under reducing conditions.
Predicted band size: 15 kDa
Observed band size: 17 kDa why is the actual band size different from the predicted?
Exposure time: 2 minutes -
Western blot - Anti-Histone H3 (di methyl K4) antibody - ChIP Grade (ab11946)Lane 1 : Marker
Lanes 2-12 : Anti-Histone H3 (di methyl K4) antibody - ChIP Grade (ab11946) at 1 µg/ml
Lane 2 : Calf Thymus Histone Preparation Nuclear Lysate (ab121) at 0.5 µg
Lane 3 : Calf Thymus Histone Preparation Nuclear Lysate (ab121) at 0.5 µg withHuman Histone H3 (unmodified ) peptide (ab7228) at 0.75 µg/ml
Lane 4 : Calf Thymus Histone Preparation Nuclear Lysate (ab121) at 0.5 µg withHuman Histone H3 (mono methyl K4) peptide (ab1340) at 0.75 µg/ml
Lane 5 : Calf Thymus Histone Preparation Nuclear Lysate (ab121) at 0.5 µg withHuman Histone H3 (di methyl K4) peptide (ab7768) at 0.75 µg/ml
Lane 6 : Calf Thymus Histone Preparation Nuclear Lysate (ab121) at 0.5 µg withHuman Histone H3 (tri methyl K4) peptide (ab1342) at 0.75 µg/ml
Lane 7 : Calf Thymus Histone Preparation Nuclear Lysate (ab121) at 0.5 µg withHuman Histone H3 (mono methyl K9) peptide (ab1771) at 0.75 µg/ml
Lane 8 : Calf Thymus Histone Preparation Nuclear Lysate (ab121) at 0.5 µg withHuman Histone H3 (di methyl K9) peptide (ab1772) at 0.75 µg/ml
Lane 9 : Calf Thymus Histone Preparation Nuclear Lysate (ab121) at 0.5 µg withHuman Histone H3 (tri methyl K9) peptide (ab1773) at 0.75 µg/ml
Lane 10 : Calf Thymus Histone Preparation Nuclear Lysate (ab121) at 0.5 µg withHuman Histone H3 (mono methyl K27) peptide (ab1780) at 0.75 µg/ml
Lane 11 : Calf Thymus Histone Preparation Nuclear Lysate (ab121) at 0.5 µg withHuman Histone H3 (di methyl K27) peptide (ab1781) at 0.75 µg/ml
Lane 12 : Calf Thymus Histone Preparation Nuclear Lysate (ab121) at 0.5 µg withHuman Histone H3 (tri methyl K27) peptide (ab1782) at 0.75 µg/ml
Secondary
Lanes 2-12 : Rabbit polyclonal to Goat IgG (Alexa Fluor® 680) 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? -
Western blot - Anti-Histone H3 (di methyl K4) antibody - ChIP Grade (ab11946)Anti-Histone H3 (di methyl K4) antibody - ChIP Grade (ab11946) at 1 µg/ml + PC12 (Rat adrenal pheochromocytoma cell line) Whole Cell Lysate at 10 µg
Secondary
Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (ab97080) at 1/5000 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?
Exposure time: 20 minutes -
Immunocytochemistry/ Immunofluorescence - Anti-Histone H3 (di methyl K4) antibody - ChIP Grade (ab11946)Image courtesy of an Abreview submitted by Dr. Kirk McManus, Univ. of Manitoba/Cancer Care MICB, Canadaab11946 (1/500) staining Histone H3 (di methyl K4) in assynchronous HeLa cells (green). Cells were fixed in paraformaldehyde, permeabilized with 0.5% Triton X100 and counterstained with DAPI in order to highlight the nucleus (red). For further experimental details please refer to Abreview. -
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H3 (di methyl K4) antibody - ChIP Grade (ab11946)IHC image of Histone H3 staining in human normal cervix formalin fixed paraffin embedded tissue section, performed on a Leica BondTM system using the standard protocol B. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab11946, 1µg/ml, for 15 mins at room temperature. A donkey anti-goat biotinylated secondary antibody was used to detect the primary, and visualized using an HRP conjugated ABC system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.
For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.
Datasheets and documents
References
This product has been referenced in:
- Shooshtarizadeh P et al. Gfi1b regulates the level of Wnt/ß-catenin signaling in hematopoietic stem cells and megakaryocytes. Nat Commun 10:1270 (2019). Read more (PubMed: 30894540) »
- Tan AHY et al. Lysine-Specific Histone Demethylase 1A Regulates Macrophage Polarization and Checkpoint Molecules in the Tumor Microenvironment of Triple-Negative Breast Cancer. Front Immunol 10:1351 (2019). Read more (PubMed: 31249575) »
