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RecombinantRabMAb

Recombinant Anti-Histone H3.3 (mutated G34R) antibody [EPR23519-91] - ChIP Grade (ab254402)

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ChIP - Anti-Histone H3.3 (mutated G34R) antibody [EPR23519-91] - ChIP Grade (ab254402)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H3.3 (mutated G34R) antibody [EPR23519-91] - ChIP Grade (ab254402)
  • Western blot - Anti-Histone H3.3 (mutated G34R) antibody [EPR23519-91] - ChIP Grade (ab254402)
  • Flow Cytometry (Intracellular) - Anti-Histone H3.3 (mutated G34R) antibody [EPR23519-91] - ChIP Grade (ab254402)
  • Immunoprecipitation - Anti-Histone H3.3 (mutated G34R) antibody [EPR23519-91] - ChIP Grade (ab254402)
  • Dot Blot - Anti-Histone H3.3 (mutated G34R) antibody [EPR23519-91] - ChIP Grade (ab254402)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H3.3 (mutated G34R) antibody [EPR23519-91] - ChIP Grade (ab254402)
  • Anti-Histone H3.3 (mutated G34R) antibody [EPR23519-91] - ChIP Grade (ab254402)

Key features and details

  • Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
  • Rabbit monoclonal [EPR23519-91] to Histone H3.3 (mutated G34R) - ChIP Grade
  • Suitable for: IP, WB, ChIP, Dot blot, Flow Cyt (Intra), IHC-P
  • Reacts with: Human

Conjugates logo Related conjugates and formulations

Carrier Free

Overview

  • Product name

    Anti-Histone H3.3 (mutated G34R) antibody [EPR23519-91] - ChIP Grade
    See all Histone H3.3 primary antibodies

  • Description

    Rabbit monoclonal [EPR23519-91] to Histone H3.3 (mutated G34R) - ChIP Grade

  • Host species

    Rabbit

  • Tested applications

    Suitable for: IP, WB, ChIP, Dot blot, Flow Cyt (Intra), IHC-Pmore details
    Unsuitable for: ICC/IF

  • Species reactivity

    Reacts with: Human

  • Immunogen

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

  • Positive control

    • WB: HEK-293T transfected with Histone H3.3 G34R expression vector containing a myc-His-tag®, whole cell lysate. IHC-P: Human giant tumor of bone tissue. Flow Cyt (intra): HEK-293T cells transfected with myc-tagged Histone H3.3 G34R expression vector. IP: HEK-293T transfected with Histone H3.3 G34R expression vector containing a myc-His-tag® whole cell lysate. ChIP: Chromatin prepared from HEK-293T transfected with Histone H3.3 (mutated G34 R).

  • 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

Applications

The Abpromise guarantee

Our Abpromise guarantee covers the use of ab254402 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
IP
1/30.
WB
1/1000. Detects a band of approximately 20 kDa (predicted molecular weight: 15 kDa).
ChIP
Use 2 µg for 25 µg of chromatin.
Dot blot
1/1000.
Flow Cyt (Intra)
1/5000.
IHC-P
1/2000. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
Notes
IP
1/30.
WB
1/1000. Detects a band of approximately 20 kDa (predicted molecular weight: 15 kDa).
ChIP
Use 2 µg for 25 µg of chromatin.
Dot blot
1/1000.
Flow Cyt (Intra)
1/5000.
IHC-P
1/2000. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
Application notes
Is unsuitable for ICC/IF.

Target

  • Function

    Variant histone H3 which replaces conventional H3 in a wide range of nucleosomes in active genes. Constitutes the predominant form of histone H3 in non-dividing cells and is incorporated into chromatin independently of DNA synthesis. Deposited at sites of nucleosomal displacement throughout transcribed genes, suggesting that it represents an epigenetic imprint of transcriptionally active chromatin. 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 throughout the cell cycle independently of DNA synthesis.

  • 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.
    Specifically enriched in modifications associated with active chromatin such as methylation at Lys-5 (H3K4me), Lys-37 and Lys-80. 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), which are linked to gene repression, are underrepresented. 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. Phosphorylation on Ser-32 (H3S31ph) is specific to regions bordering centromeres in metaphase chromosomes.
    Ubiquitinated. Monoubiquitinated by RAG1 in lymphoid cells, monoubiquitination is required for V(D)J recombination.

  • Cellular localization

    Nucleus. Chromosome.
  • Information by UniProt

  • Database links

    • Alternative names

      • H3 histone family 3A antibody
      • H3 histone family 3B antibody
      • H3 histone, family 3B (H3.3B) antibody
      • H3.3 antibody
      • H3.3A antibody
      • H3.3B antibody
      • H33_HUMAN antibody
      • H3F3 antibody
      • H3F3A antibody
      • H3f3b antibody
      • Histone H3.3 antibody
      • Histone H3.3Q antibody
      • Histone H3.A antibody
      • Histone H3.B antibody
      • MGC87782 antibody
      • MGC87783 antibody
      see all

    Images

    • ChIP - Anti-Histone H3.3 (mutated G34R) antibody [EPR23519-91] - ChIP Grade (ab254402)
      ChIP - Anti-Histone H3.3 (mutated G34R) antibody [EPR23519-91] - ChIP Grade (ab254402)

      Chromatin was prepared from HEK-293T transfected with Histone H3.3(mutated G34 R) and HEK-293T transfacted with Histone H3.3(WT) 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 ab254402 (red), or 2 µg of rabbit normal IgG ab172730 (gray) and 25 µl of Protein A/G Dynabeads. The immunoprecipitated DNA was quantified by real time PCR (Taqman approach for active and inactive loci).

      *https://www.abcam.com/resources?keywords=X%20ChIP%20protocol

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H3.3 (mutated G34R) antibody [EPR23519-91] - ChIP Grade (ab254402)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H3.3 (mutated G34R) antibody [EPR23519-91] - ChIP Grade (ab254402)

      Immunohistochemical analysis of paraffin-embedded human giant cell tumor of bone tissue labeling Histone H3.3 (mutated G34 R) with ab254402 at 1/2000 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear staining on human giant cell tumor of bone (PMID: 29241742). The section was incubated with ab254402 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin.

      Secondary antibody only control: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

      Heat mediated antigen retrieval with Citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins

    • Western blot - Anti-Histone H3.3 (mutated G34R) antibody [EPR23519-91] - ChIP Grade (ab254402)
      Western blot - Anti-Histone H3.3 (mutated G34R) antibody [EPR23519-91] - ChIP Grade (ab254402)
      All lanes : Anti-Histone H3.3 (mutated G34R) antibody [EPR23519-91] - ChIP Grade (ab254402) at 1/1000 dilution

      Lane 1 : HEK-293T (human embryonic kidney) transfected with Histone H3.3 G34R expression vector containi a myc-His-tag®, whole cell lysate
      Lane 2 : HEK-293T transfected with Histone H3.3 (WT) expression vector containing a myc-His-tag®, whole cell lysate
      Lane 3 : HEK-293T (human embryonic kidney) transfected with Histone H3.3 G34V expression vector containing a myc-His-tag®, whole cell lysate
      Lane 4 : HEK-293T (human embryonic kidney) transfected with Histone H3.3 G34W expression vector containngi a myc-His-tag®, whole cell lysate

      Lysates/proteins at 20 µg per lane.

      Secondary
      All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/100000 dilution

      Predicted band size: 15 kDa
      Observed band size: 20 kDa why is the actual band size different from the predicted?



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

      Exposure time: 26 seconds. 

    • Flow Cytometry (Intracellular) - Anti-Histone H3.3 (mutated G34R) antibody [EPR23519-91] - ChIP Grade (ab254402)
      Flow Cytometry (Intracellular) - Anti-Histone H3.3 (mutated G34R) antibody [EPR23519-91] - ChIP Grade (ab254402)

      Intracellular flow cytometric analysis of 4% paraformaldehyde fixed, 90% methanol permeabilized HEK-293T (Human embryonic kidney epithelial cell) (transfected with myc-tagged Histone H3.3 WT expression vector) (Left) or myc-tagged Histone H3.3 G34R expression vector (Right) cells labelling Histone H3.3 (mutated G34 R) with ab254402 at 1/5000 dilution (0.01ug) (Both panels). A Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) at 1/2000 dilution was used as the secondary antibody. 

       

       

    • Immunoprecipitation - Anti-Histone H3.3 (mutated G34R) antibody [EPR23519-91] - ChIP Grade (ab254402)
      Immunoprecipitation - Anti-Histone H3.3 (mutated G34R) antibody [EPR23519-91] - ChIP Grade (ab254402)

      Histone H3.3 (mutated G34 R) was immunoprecipitated from 0.35 mg HEK-293T (human embryonic kidney) (transfected with Histone H3.3 G34R expression vector containing a myc-His-tag®) whole cell lysate with ab254402 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab254402 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

      Lane 1: HEK-293T (human embryonic kidney) transfected with Histone H3.3 G34R expression vector containing a myc-His-tag®, whole cell lysate 10 ug

      Lane 2: ab254402 IP in HEK-293T transfected with Histone H3.3 G34R expression vector containing a myc-His-tag® whole cell lysate

      Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab254402 in HEK-293T transfected with Histone H3.3 G34R expression vector containing a myc-His-tag® whole cell lysate

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

      Exposure time: 41 seconds.

       

    • Dot Blot - Anti-Histone H3.3 (mutated G34R) antibody [EPR23519-91] - ChIP Grade (ab254402)
      Dot Blot - Anti-Histone H3.3 (mutated G34R) antibody [EPR23519-91] - ChIP Grade (ab254402)

      Dot blot analysis of Histone H3.3 (mutated G34 R) labeled with ab254402 at 1/1000 dilution.

      Lane 1: Histone H3.3 H3G34R peptide (aa28-40).
      Lane 2: Histone H3.3 H3G34R peptide (aa26-36).
      Lane 3: Histone H3.3 WT peptide (aa26-40).

      Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution was used as secondary antibody.

      Blocking and dilution buffer: 5% NFDM/TBST.

      Exposure time: 3 minutes.

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H3.3 (mutated G34R) antibody [EPR23519-91] - ChIP Grade (ab254402)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H3.3 (mutated G34R) antibody [EPR23519-91] - ChIP Grade (ab254402)

      Immunohistochemical analysis of paraffin-embedded human testis tissue labeling Histone H3.3 (mutated G34 R) with ab254402 at 1/2000 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Negative control: no staining on human testis. The section was incubated with ab254402 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin.

      Secondary antibody only control: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

      Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

    • Anti-Histone H3.3 (mutated G34R) antibody [EPR23519-91] - ChIP Grade (ab254402)
      Anti-Histone H3.3 (mutated G34R) antibody [EPR23519-91] - ChIP Grade (ab254402)

    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.

    Click here to view the general protocols

    References (0)

    Publishing research using ab254402? Please let us know so that we can cite the reference in this datasheet.

    ab254402 has not yet been referenced specifically in any publications.

    Customer reviews and Q&As

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    IHC - Wholemount abreview for Anti-Histone H3.3 (mutated G34R) antibody [EPR23519-91] - ChIP Grade

     Inconclusive
    Abreviews
    Application
    IHC - Wholemount
    Sample
    Mouse Tissue (Brain)
    Specification
    Brain
    Read More
    The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

    MISS. Aalaa Abdallah

    Verified customer

    Submitted Apr 02 2021

    Please note: All products are "FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES"
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