RecombinantRabMAb

Recombinant Anti-Histone H3 (mutated K27M) antibody [EPR18340] - ChIP Grade (ab190631)

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ChIP - Anti-Histone H3 (mutated K27M) antibody [EPR18340] - ChIP Grade (ab190631)
  • Western blot - Anti-Histone H3 (mutated K27M) antibody [EPR18340] - ChIP Grade (ab190631)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H3 (mutated K27M) antibody [EPR18340] - ChIP Grade (ab190631)
  • Western blot - Anti-Histone H3 (mutated K27M) antibody [EPR18340] - ChIP Grade (ab190631)
  • Indirect ELISA - Anti-Histone H3 (mutated K27M) antibody [EPR18340] - ChIP Grade (ab190631)
  • Immunoprecipitation - Anti-Histone H3 (mutated K27M) antibody [EPR18340] - ChIP Grade (ab190631)
  • Immunocytochemistry/ Immunofluorescence - Anti-Histone H3 (mutated K27M) antibody [EPR18340] - ChIP Grade (ab190631)
  • Western blot - Anti-Histone H3 (mutated K27M) antibody [EPR18340] - ChIP Grade (ab190631)
  • 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, Recombinant fragment

Conjugates logo Related conjugates and formulations

Carrier Free

Overview

  • 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, Recombinant fragment

  • Immunogen

    Synthetic peptide. This information is proprietary to Abcam and/or its suppliers.

  • 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
    For more information see here.

    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

  • 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...

  • Purity

    Protein A purified

  • Clonality

    Monoclonal

  • Clone number

    EPR18340

  • Isotype

    IgG

  • Research areas

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
WB (2)
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)
1/500.
ICC/IF (1)
1/5000.
IP
1/30.
ChIP
Use 2 µg for 25 µg of chromatin.
Notes
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

  • 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 (mutated K27M) antibody [EPR18340] - ChIP Grade (ab190631)
    ChIP - Anti-Histone H3 (mutated K27M) antibody [EPR18340] - ChIP Grade (ab190631)

    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

  • Western blot - Anti-Histone H3 (mutated K27M) antibody [EPR18340] - ChIP Grade (ab190631)
    Western blot - Anti-Histone H3 (mutated K27M) antibody [EPR18340] - ChIP Grade (ab190631)
    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.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H3 (mutated K27M) antibody [EPR18340] - ChIP Grade (ab190631)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H3 (mutated K27M) antibody [EPR18340] - ChIP Grade (ab190631)This image is courtesy of Dr Yusuke Tomita, Northwestern University.
    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.

     

  • Western blot - Anti-Histone H3 (mutated K27M) antibody [EPR18340] - ChIP Grade (ab190631)
    Western blot - Anti-Histone H3 (mutated K27M) antibody [EPR18340] - ChIP Grade (ab190631)
    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 seconds


    Blocking/Diluting buffer and concentration: 5% NFDM/TBST.

  • Indirect ELISA - Anti-Histone H3 (mutated K27M) antibody [EPR18340] - ChIP Grade (ab190631)
    Indirect ELISA - Anti-Histone H3 (mutated K27M) antibody [EPR18340] - ChIP Grade (ab190631)

    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.

  • Immunoprecipitation - Anti-Histone H3 (mutated K27M) antibody [EPR18340] - ChIP Grade (ab190631)
    Immunoprecipitation - Anti-Histone H3 (mutated K27M) antibody [EPR18340] - ChIP Grade (ab190631)

    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

     

  • Immunocytochemistry/ Immunofluorescence - Anti-Histone H3 (mutated K27M) antibody [EPR18340] - ChIP Grade (ab190631)
    Immunocytochemistry/ Immunofluorescence - Anti-Histone H3 (mutated K27M) antibody [EPR18340] - ChIP Grade (ab190631)

    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.

  • Western blot - Anti-Histone H3 (mutated K27M) antibody [EPR18340] - ChIP Grade (ab190631)
    Western blot - Anti-Histone H3 (mutated K27M) antibody [EPR18340] - ChIP Grade (ab190631)
    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 seconds


    Blocking/Dilution buffer: 5% BSA/TBST.

  • Anti-Histone H3 (mutated K27M) antibody [EPR18340] - ChIP Grade (ab190631)
    Anti-Histone H3 (mutated K27M) antibody [EPR18340] - ChIP Grade (ab190631)

References (2)

Publishing research using ab190631? Please let us know so that we can cite the reference in this datasheet.

ab190631 has been referenced in 2 publications.

  • 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
  • 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

Customer reviews and Q&As

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1-4 of 4 Abreviews or Q&A

Western blot abreview for Anti-Histone H3 (mutated K27M) antibody [EPR18340] - ChIP Grade

 Excellent
Abreviews
abreview image
Application
Western blot
Sample
Human Cell lysate - whole cell (Neural stem cells and DIPG cells)
Gel Running Conditions
Reduced Denaturing (Novex 10-20% Tricine Gel)
Loading amount
11 µg
Specification
Neural stem cells and DIPG cells
Blocking step
Intercept (PBS) Blocking Buffer as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 50% · Temperature: ~22°C
Read More

Abcam user community

Verified customer

Submitted Jan 19 2021

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) abreview for Anti-Histone H3 (mutated K27M) antibody [EPR18340] - ChIP Grade

 Excellent
Abreviews
abreview image
Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Mouse Tissue sections (Brain tumor with H3K27M mutation)
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: Cell Conditioning 1
Permeabilization
No
Specification
Brain tumor with H3K27M mutation
Fixative
Neutral Buffer Formalin
Read More

Dr. Yusuke Tomita

Verified customer

Submitted Aug 27 2020

Immunocytochemistry/ Immunofluorescence abreview for Anti-Histone H3 (mutated K27M) antibody [EPR18340] - ChIP Grade

 Excellent
Abreviews
abreview image
Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (Adult and Pediatric Glioblastoma Primary Cell line)
Permeabilization
Yes - 0.5% Triton-X-100 in PBS
Specification
Adult and Pediatric Glioblastoma Primary Cell line
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: 18°C
Fixative
Formaldehyde
Read More

Tatiana Kaptzan

Verified customer

Submitted Feb 23 2016

Western blot abreview for Anti-Histone H3 (mutated K27M) antibody [EPR18340] - ChIP Grade

 Excellent
Abreviews
abreview image
Application
Western blot
Sample
Human Cell lysate - whole cell (Glioblastoma primary cell lines)
Gel Running Conditions
Non-reduced Denaturing (12.5% Criterion Tris-HCl Gel)
Loading amount
20 µg
Specification
Glioblastoma primary cell lines
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 18°C
Read More

Tatiana Kaptzan

Verified customer

Submitted Feb 23 2016

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