Recombinant Anti-Histone H3 (di methyl K4) antibody [Y47] - ChIP Grade (ab32356)
Key features and details
- Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
- Rabbit monoclonal [Y47] to Histone H3 (di methyl K4) - ChIP Grade
- Suitable for: ChIP, ChIP-sequencing, WB, IHC-P, ICC/IF, Flow Cyt (Intra), IP
- Reacts with: Mouse, Rat, Chicken, Cow, Human, African green monkey
Related conjugates and formulations
Overview
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Product name
Anti-Histone H3 (di methyl K4) antibody [Y47] - ChIP Grade
See all Histone H3 primary antibodies -
Description
Rabbit monoclonal [Y47] to Histone H3 (di methyl K4) - ChIP Grade -
Host species
Rabbit -
Specificity
This antibody only detects Histone H3 dimethylated on Lysine 4. -
Tested applications
Suitable for: ChIP, ChIP-sequencing, WB, IHC-P, ICC/IF, Flow Cyt (Intra), IPmore details -
Species reactivity
Reacts with: Mouse, Rat, Chicken, Cow, Human, African green monkey
Predicted to work with: Sheep, Saccharomyces cerevisiae, Xenopus laevis, Arabidopsis thaliana, Monkey, Zebrafish, Mammals, Rice -
Immunogen
Synthetic peptide within Human Histone H3 aa 1-100 (di methyl K4). The exact sequence is proprietary.
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Positive control
- WB: HeLa, HEK-293, SH-SY5Y, C6 L6, NIH/3T3, COS-1, UMNSAH/DF-1 and MDBK cell lysates. ICC/IF: HepG2 cells. Flow Cyt (intra): HeLa and HepG2 cells. IHC-P: Human cervical carcinoma, mouse colon and rat spleen tissues. ChIP: Chromatin prepared from Hela cells, ALDO ChIP primer pair ab269260. ChIP-seq: Chromatin prepared from Hela cells. IP: Hep-G2 cells.
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General notes
Every new batch of this antibody is tested in house in ChIP.
This product is a recombinant monoclonal antibody, which offers several advantages including:
- - High batch-to-batch consistency and reproducibility
- - Improved sensitivity and specificity
- - Long-term security of supply
- - Animal-free production
Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb® patents.
We are constantly working hard to ensure we provide our customers with best in class antibodies. As a result of this work we are pleased to now offer this antibody in purified format. We are in the process of updating our datasheets. The purified format is designated 'PUR' on our product labels. If you have any questions regarding this update, please contact our Scientific Support team.
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. Avoid freeze / thaw cycle. -
Storage buffer
pH: 7.20
Preservative: 0.01% Sodium azide
Constituents: 59% PBS, 40% Glycerol, 0.05% BSA -
Concentration information loading...
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Purity
Protein A purified -
Clonality
Monoclonal -
Clone number
Y47 -
Isotype
IgG -
Research areas
Associated products
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Alternative Versions
- Anti-Histone H3 (di methyl K4) antibody [Y47] - BSA and Azide free (ab173324)
- Alexa Fluor® 488 Anti-Histone H3 (di methyl K4) antibody [Y47] (ab200024)
- Alexa Fluor® 647 Anti-Histone H3 (di methyl K4) antibody [Y47] (ab200026)
- Alexa Fluor® 594 Anti-Histone H3 (di methyl K4) antibody [Y47] (ab207864)
- Alexa Fluor® 405 Anti-Histone H3 (di methyl K4) antibody [Y47] (ab207865)
- PE Anti-Histone H3 (di methyl K4) antibody [Y47] (ab208741)
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ChIP Related Products
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Compatible Secondaries
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Isotype control
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Positive Controls
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Recombinant Protein
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Related Products
Applications
The Abpromise guarantee
Our Abpromise guarantee covers the use of ab32356 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 |
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ChIP | (2) |
Use 2 µg for 25 µg of chromatin.
Use ALDOA ChIP primer pair ab269260 as positive control. |
ChIP-sequencing |
Use 4 µg for 30 µg of chromatin.
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WB | (3) |
1/2000. Detects a band of approximately 17 kDa (predicted molecular weight: 15 kDa).
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IHC-P | (1) |
1/800. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
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ICC/IF | (4) |
1/1000.
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Flow Cyt (Intra) |
1/20 - 1/100.
ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody. |
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IP |
1/30.
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Notes |
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ChIP
Use 2 µg for 25 µg of chromatin. Use ALDOA ChIP primer pair ab269260 as positive control. |
ChIP-sequencing
Use 4 µg for 30 µg of chromatin. |
WB
1/2000. Detects a band of approximately 17 kDa (predicted molecular weight: 15 kDa). |
IHC-P
1/800. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol. |
ICC/IF
1/1000. |
Flow Cyt (Intra)
1/20 - 1/100. ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody. |
IP
1/30. |
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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Chromatin was prepared from HeLa cells. Cells were fixed with 1% formaldehyde for 10 minutes. ChIP was performed with 107 cells and 4 µg of Anti-Histone H3 (di methyl K4) antibody [Y47] - ChIP Grade (ab32356). ChIP DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 30 million reads.
Additional screenshots of mapped reads can be downloaded here.
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Chromatin was prepared from HeLa cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10 minutes. The ChIP was performed with 25µg of chromatin, 2µg of ab32356 (red), and 20µl of Anti Rabbit IgG sepharose beads. 2μg of rabbit normal IgG was added to the beads control (grey). The immunoprecipitated DNA was quantified by real time PCR (Sybr green approach). Primers and probes are located in the first kb of the transcribed region.
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Chromatin was prepared from HeLa cells. Cells were fixed with 1% formaldehyde for 10 minutes. ChIP was performed with 30 µg of chromatin and 4 µg of Anti-Histone H3 (di methyl K4) antibody [Y47] - ChIP Grade (ab32356). ChIP DNA was sequenced on the Illumina NextSeq 500 to a depth of 30 million reads. ChIP-Seq validation performed by Active Motif, Carlsbad, CA.
Additional screenshots of mapped reads can be downloaded here.
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ab32356 (purified) at 1/30 dilution (20 µg/ml) immunoprecipitating Histone H3 di methyl K4 in HepG2 whole cell lysate.
Lane 1 (input): HepG2 (human hepatocellular carcinoma epithelial cell) whole cell lysate 10µg
Lane 2 (+): ab32356 & HepG2 whole cell lysate
Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab32356 in HepG2 whole cell lysate
For western blotting, ab32356 at 1/500 and veriBlot for IP secondary antibody (HRP) (ab131366) was used at 1/1000 dilution.
Blocking and diluting buffer: 5% NFDM /TBST. -
Flow Cytometry (Intracellular) - Anti-Histone H3 (di methyl K4) antibody [Y47] - ChIP Grade (ab32356)
Intracellular Flow Cytometry analysis of HepG2 (human hepatocellular carcinomal) cells labeling Histone H3 di methyl K4 with purified ab32356 at 1/500 dilution (1.17µg/ml) (Red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. A Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) secondary antibody was used at 1/2000 dilution. Isotype control - Rabbit monoclonal IgG (ab172730) / Black. Unlabeled control - Unlabelled cells / blue.
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All lanes : Anti-Histone H3 (di methyl K4) antibody [Y47] - ChIP Grade (ab32356) at 1/10000 dilution (purified)
Lane 1 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate
Lane 2 : HEK-293 (Human epithelial cell line from embryonic kidney) whole cell lysate
Lane 3 : SH-SY5Y (Human neuroblastoma cell line from bone marrow) whole cell lysate
Lane 4 : C6 (Rat glial tumor cell line) whole cell lysate
Lysates/proteins at 20 µg per lane.
Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution
Predicted band size: 15 kDa
Additional bands at: 17 kDa. We are unsure as to the identity of these extra bands.
Exposure time: 5 seconds -
Immunocytochemistry/ Immunofluorescence - Anti-Histone H3 (di methyl K4) antibody [Y47] - ChIP Grade (ab32356)
Immunocytochemistry/Immunofluorescence analysis of HepG2 cells labelling Histone H3 (di methyl K4) with purified ab32356 at a dilution of 1/1000. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. ab150077, an Alexa Fluor® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody. The cells were co-stained with ab7291, a mouse anti-tubulin (1/1000) using ab150120, an Alexa Fluor® 594-conjugated goat anti-mouse IgG (1/1000) as the secondary antibody. Nuclei counterstained with DAPI (blue).
Control 1: primary antibody (1/1000) and secondary antibody, ab150120, an Alexa Fluor® 594-conjugated goat anti-mouse IgG (1/1000).
Control 2: ab7291 (1/1000) and secondary antibody, ab150077, an Alexa Fluor® 488-conjugated goat anti-rabbit IgG (1/1000).
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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H3 (di methyl K4) antibody [Y47] - ChIP Grade (ab32356)
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human cervical carcinoma tissue labelling Histone H3 (di methyl K4) with purified ab32356 at a dilution of 1/800. Antigen retrieval was performed using Tris/EDTA buffer, pH9. ab97051, a HRP-conjugated goat anti-rabbit IgG H&L was used as the secondary antibody (1/500). Counter stained with hematoxylin.
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Flow Cytometry (Intracellular) - Anti-Histone H3 (di methyl K4) antibody [Y47] - ChIP Grade (ab32356)
Intracellular Flow Cytometry analysis of HeLa cells labelling Histone H3 (di methyl K4) with purified ab32356 at 1/150 (red). Cells were fixed with 80% methanol. An Alexa Fluorr® 488-conjugated goat anti-rabbit IgG (1/500) was used as the secondary antibody. Black - Isotype control, rabbit monoclonal IgG (ab172730). Blue - Unlabelled control, cells without incubation with primary and secondary antibodies.
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All lanes : Anti-Histone H3 (di methyl K4) antibody [Y47] - ChIP Grade (ab32356) at 1/2000 dilution (purified)
Lane 1 : L6 (Rat skeletal muscle cell line) whole cell lysate
Lane 2 : NIH/3T3 (Mouse embryonic fibroblast cell line) whole cell lysate
Lane 3 : COS-1 (African green monkey kidney fibroblast-like cell line) whole cell lysate
Lane 4 : UMNSAH/DF-1 (Transformed chicken embryonic fibroblast cell line) whole cell lysate
Lane 5 : MDBK (Bovine kidney cell line) whole cell lysate
Lysates/proteins at 20 µg per lane.
Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/1000 dilution
Predicted band size: 15 kDa
Observed band size: 17 kDa why is the actual band size different from the predicted?
Exposure time: 5 seconds -
Immunocytochemistry/ Immunofluorescence - Anti-Histone H3 (di methyl K4) antibody [Y47] - ChIP Grade (ab32356)
ICC/IF image of unpurified ab32356 stained HepG2 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 (ab32356 , 5µg/ml) overnight at +4°C. The secondary antibody (green) was a goat anti-rabbit DyLight® 488 (IgG - H&L, pre-adsorbed) (ab96899) used at a 1/250 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.
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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H3 (di methyl K4) antibody [Y47] - ChIP Grade (ab32356)
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of mouse colon tissue labelling Histone H3 (di methyl K4) with purified ab32356 at a dilution of 1/800. Antigen retrieval was performed using Tris/EDTA buffer, pH9. ab97051, a HRP-conjugated goat anti-rabbit IgG H&L was used as the secondary antibody (1/500). Counter stained with hematoxylin.
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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H3 (di methyl K4) antibody [Y47] - ChIP Grade (ab32356)
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of rat spleen tissue labelling Histone H3 (di methyl K4) with purified ab32356 at a dilution of 1/800. Antigen retrieval was performed using Tris/EDTA buffer, pH9. ab97051, a HRP-conjugated goat anti-rabbit IgG H&L was used as the secondary antibody (1/500). Counter stained with hematoxylin.
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Flow Cytometry (Intracellular) - Anti-Histone H3 (di methyl K4) antibody [Y47] - ChIP Grade (ab32356)
Overlay histogram showing HeLa cells stained with unpurified ab32356 (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 (ab32356, 1/100 dilution) for 30 min at 22°C. The secondary antibody used was a goat anti-rabbit DyLight® 488 (IgG; H+L) (ab96899) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed.
Protocols
To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.
Datasheets and documents
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SDS download
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Datasheet download
References (171)
ab32356 has been referenced in 171 publications.
- Tresenrider A et al. Integrated genomic analysis reveals key features of long undecoded transcript isoform-based gene repression. Mol Cell 81:2231-2245.e11 (2021). PubMed: 33826921
- Vonk PJ & Ohm RA H3K4me2 ChIP-Seq reveals the epigenetic landscape during mushroom formation and novel developmental regulators of Schizophyllum commune. Sci Rep 11:8178 (2021). PubMed: 33854169
- Zhou M et al. miR-181d/RBP2/NF-?B p65 Feedback Regulation Promotes Chronic Myeloid Leukemia Blast Crisis. Front Oncol 11:654411 (2021). PubMed: 33842368
- Theisen ER et al. Chromatin profiling reveals relocalization of lysine-specific demethylase 1 by an oncogenic fusion protein. Epigenetics 16:405-424 (2021). PubMed: 32842875
- Redl S et al. Extensive nuclear gyration and pervasive non-genic transcription during primordial germ cell development in zebrafish. Development 148:N/A (2021). PubMed: 33298460