Recombinant Anti-Histone H3 (mutated K27M) antibody [EPR18340] - ChIP Grade (ab190631)
Key features and details
- Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
- Rabbit monoclonal [EPR18340] to Histone H3 (mutated K27M) - ChIP Grade
- Suitable for: WB, Indirect ELISA, IHC-P, ICC/IF, IP, ChIP
- Reacts with: Mouse, Human
Related conjugates and formulations
Overview
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Product name
Anti-Histone H3 (mutated K27M) antibody [EPR18340] - ChIP Grade
See all Histone H3 primary antibodies -
Description
Rabbit monoclonal [EPR18340] to Histone H3 (mutated K27M) - ChIP Grade -
Host species
Rabbit -
Tested applications
Suitable for: WB, Indirect ELISA, IHC-P, ICC/IF, IP, ChIPmore details -
Species reactivity
Reacts with: Mouse, Human -
Immunogen
Synthetic peptide. This information is proprietary to Abcam and/or its suppliers.
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General notes
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.
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 long term. Avoid freeze / thaw cycle. -
Storage buffer
pH: 7.2
Preservative: 0.01% Sodium azide
Constituents: PBS, 40% Glycerol, 0.05% BSA -
Concentration information loading...
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Purity
Protein A purified -
Clonality
Monoclonal -
Clone number
EPR18340 -
Isotype
IgG -
Research areas
Associated products
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Alternative Versions
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Compatible Secondaries
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Isotype control
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Recombinant Protein
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Related Products
Applications
The Abpromise guarantee
Our Abpromise guarantee covers the use of ab190631 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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WB | (2) |
1/1000. Detects a band of approximately 15 kDa (predicted molecular weight: 15 kDa).
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Indirect ELISA |
Use a concentration of 1 µg/ml.
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IHC-P | (1) |
1/500.
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ICC/IF | (1) |
1/5000.
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IP |
1/30.
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ChIP |
Use 2 µg for 25 µg of chromatin.
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Notes |
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WB
1/1000. Detects a band of approximately 15 kDa (predicted molecular weight: 15 kDa). |
Indirect ELISA
Use a concentration of 1 µg/ml. |
IHC-P
1/500. |
ICC/IF
1/5000. |
IP
1/30. |
ChIP
Use 2 µg for 25 µg of chromatin. |
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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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of tissue sections from(A) HEK-293T transfected with Histone H3 (mutated K27M) expression vector containing a his tag. (B) HEK-293T transfected with wildtype Histone H3 expression vector containing a his tag. (C) HEK-293T transfected with empty vector containing a his tag, labeling Histone H3 (mutated K27M) with ab190631 at 1/1000 dilution and ready to use secondary LeicaDS9800 (Bond™ Polymer Refine Detection) counterstained with Hematoxylin.
Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins
Positive staining on HEK-293T transfected with Histone H3 (mutated K27M) expression vector cell pellets (image A), no staining on HEK-293T transfected with wildtype Histone H3 expression vector cell pellets (Image B) or empty vector cell pellets (Image C).
The section was incubated with ab190631 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument -
Chromatin was prepared from HEK-293T transfected with myc-His tagged Histone H3 (K27M) and Histone H3 WT cells according to the Abcam Dual-X-ChIP protocol*. Cells were fixed with 1.5 mM EGS for 30mins and then formaldehyde for 10min.
The ChIP was performed with 25 µg of chromatin, 2 µg of ab190631 (red), 2 µg of ab213204 (red) (bottom panel, served as internal control) or 2 µg of rabbit normal IgG ab172730 (gray) and 20 µl of Protein A/G sepharose beads. 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.
*https://www.abcam.com/resources?keywords=X%20ChIP%20protocol
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All lanes : Anti-Histone H3 (mutated K27M) antibody [EPR18340] - ChIP Grade (ab190631) at 1/1000 dilution
Lane 1 : HEK-293 transfected with Histone H3.1 (K27M) expression vector containing a myc-His-tag, whole cell lysate
Lane 2 : HEK-293 transfected with Histone H3(WT) expression vector containing a myc-His-tag, whole cell lysate
Lysates/proteins at 10 µg per lane.
Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution
Predicted band size: 15 kDa
Observed band size: 18 kDa why is the actual band size different from the predicted?Blocking and diluting buffer and concentration: 5% NFDM/TBST.
Exposure time: 6 seconds.
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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of tissue sections from mouse brain tumor with and without H3K27M mutation labeling Histone H3 (mutated K27M) with ab190631 at 1/500 dilution (1 hour at 36°C in 2% BSA in PBS).Tissue was fixed with 20% Neutral Buffer Formalin for 48 hours, then changed to 70% ethanol for a week. Paraffin-embedded tissue was cut in 5 micrometer thickness with microtome. Antigen retrieval was done with Cell-Conditioning 1 (Roche) at 95 degree. A HRP conjugated monoclonal antibody (DISCOVERY UltraMap anti-Rb HRP (RUO)) was used as the secondary antibody.
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Anti-Histone H3 (mutated K27M) antibody [EPR18340] - ChIP Grade (ab190631) at 1/1000 dilution + HEK-293 transfected with Histone H3.3 (K27M) expression vector containing a myc-His-tag®, whole cell lysate at 10 µg
Secondary
Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution
Predicted band size: 15 kDa
Observed band size: 18 kDa why is the actual band size different from the predicted?
Exposure time: 3 secondsBlocking/Diluting buffer and concentration: 5% NFDM/TBST.
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ELISA using ab190631 at varying antibody concentrations and antigen concentration at 1000 ng/mL. An Alkaline Phosphatase-conjugated Goat Anti-Rabbit IgG (H+L) (1/2500) was used as the secondary antibody.
The blue line indicates binding to the Histone H3 (mutated K27M) peptide. Binding to the following peptides was not seen:
Histone H3 WT,
Histone H3 (mono methyl K27),
Histone H3 (di methyl K27),
Histone H3 (tri methyl K27).This indicates the specificity of ab190631 for mutated K27M of Histone H3.
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Histone H3 (K27 mutated to M) was immunoprecipitated from 0.35 mg HEK-293T (human embryonic kidney epithelial cell) cells transfected with myc-tagged H3(K27M) expression vector whole cell lysate 10ug with ab190631 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab190631 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1/5000 dilution.
Lane 1: HEK-293T (human embryonic kidney epithelial cell) cells transfected with myc-tagged H3(K27M) expression vector whole cell lysate 10ug
Lane 2: ab190631 IP in HEK-293T cells transfected with myc-tagged H3(K27M) expression vector whole cell lysate
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab190631 in HEK-293T cells transfected with myc-tagged H3(K27M) expression vector whole cell lysate
Blocking and dilution buffer and concentration: 5% NFDM/TBST.
Exposure time: 5 seconds
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Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HEK-293T (human embryonic kidney epithelial cell) cells labeling Histone H3 (K27 mutated to M) with ab190631 at 1/5000 (0.2 ug/ml) dilution, followed by ab150077 AlexaFluor®488 Goat anti-Rabbit secondary antibody at 1/1000 (2 ug/ml) dilution (Green). Confocal image showing nuclear staining in HEK-293T cells transfected with myc-tagged H3 (K27 mutated to M) expression vector is observed. ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 dilution (Red). The Nuclear counterstain was DAPI (Blue).
Secondary antibody only control: Secondary antibody is Ab190631 anti- H3(K27 mutated to M) ab150077 AlexaFluor®488 Goat anti-Rabbit secondary at 1/1000 (2 ug/ml) dilution.
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All lanes : Anti-Histone H3 (mutated K27M) antibody [EPR18340] - ChIP Grade (ab190631) at 1/1000 dilution
Lane 1 : His-tagged recombinant histone H3 K27M protein
Lane 2 : His-tagged recombinant wild type histone H3 protein
Lysates/proteins at 0.01 µg per lane.
Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution
Predicted band size: 15 kDa
Observed band size: 15 kDa
Exposure time: 15 secondsBlocking/Dilution buffer: 5% BSA/TBST.
Datasheets and documents
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SDS download
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Datasheet download
Certificate of Compliance
References (9)
ab190631 has been referenced in 9 publications.
- Majzner RG et al. GD2-CAR T cell therapy for H3K27M-mutated diffuse midline gliomas. Nature 603:934-941 (2022). PubMed: 35130560
- Tomita Y et al. A novel mouse model of diffuse midline glioma initiated in neonatal oligodendrocyte progenitor cells highlights cell-of-origin dependent effects of H3K27M. Glia 70:1681-1698 (2022). PubMed: 35524725
- Petrilli LL et al. Inter and intra-tumor heterogeneity of paediatric type diffuse high-grade gliomas revealed by single-cell mass cytometry. Front Oncol 12:1016343 (2022). PubMed: 36568177
- Brien GL et al. Simultaneous disruption of PRC2 and enhancer function underlies histone H3.3-K27M oncogenic activity in human hindbrain neural stem cells. Nat Genet 53:1221-1232 (2021). PubMed: 34294917
- Rakotomalala A et al. H3.3K27M Mutation Controls Cell Growth and Resistance to Therapies in Pediatric Glioma Cell Lines. Cancers (Basel) 13:N/A (2021). PubMed: 34771714
- Patel SK et al. Generation of diffuse intrinsic pontine glioma mouse models by brainstem-targeted in utero electroporation. Neuro Oncol 22:381-392 (2020). PubMed: 31638150
- An S et al. Histone tail analysis reveals H3K36me2 and H4K16ac as epigenetic signatures of diffuse intrinsic pontine glioma. J Exp Clin Cancer Res 39:261 (2020). PubMed: 33239043
- Nagaraja S et al. Histone Variant and Cell Context Determine H3K27M Reprogramming of the Enhancer Landscape and Oncogenic State. Mol Cell 76:965-980.e12 (2019). PubMed: 31588023
- Mount CW et al. Potent antitumor efficacy of anti-GD2 CAR T cells in H3-K27M+ diffuse midline gliomas. Nat Med 24:572-579 (2018). PubMed: 29662203