Recombinant
RabMAb

Anti-Histone H3.3 antibody [EPR17899] - ChIP Grade (ab176840)

Overview

  • Product name
    Anti-Histone H3.3 antibody [EPR17899] - ChIP Grade
    See all Histone H3.3 primary antibodies
  • Description
    Rabbit monoclonal [EPR17899] to Histone H3.3 - ChIP Grade
  • Host species
    Rabbit
  • Specificity
    The immunogen sequence used for this antibody is 100% identical to the protein sequence shown for H3.X and H3.Y in Szenker et al., 2011 (PubMed ID 21263457). These H3.X and H3.Y protein sequences have not been reviewed by UniProt. For further information on this antibody, please contact our technical support team.
  • Tested applications
    Suitable for: ICC/IF, ChIP, IHC-P, WB, Dot blotmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Human
  • Immunogen

    Synthetic peptide (the amino acid sequence is considered to be commercially sensitive) within Human Histone H3.3 aa 50 to the C-terminus. The exact sequence is proprietary.
    Database link: P84243

  • Positive control
    • WB: HeLa and NIH/3T3 whole cell lysates. IHC-P: Human colon, Mouse stomach and Rat colon tissues. ICC/IF: HeLa cells. ChIP: Chromatin prepared from HeLa cells.
  • General notes

     

     

    Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMab® patents

    This product is a recombinant rabbit monoclonal antibody.

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
    Preservative: 0.01% Sodium azide
    Constituents: 59% PBS, 0.05% BSA, 40% Glycerol
  • Concentration information loading...
  • Purity
    Protein A purified
  • Clonality
    Monoclonal
  • Clone number
    EPR17899
  • Isotype
    IgG
  • Research areas

Applications

Our Abpromise guarantee covers the use of ab176840 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
ICC/IF 1/1000.
ChIP Use 2 µg for 25 µg of chromatin.
IHC-P 1/1000. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
WB 1/1000. Detects a band of approximately 15 kDa (predicted molecular weight: 15 kDa).
Dot blot 1/1000.

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

  • All lanes : Anti-Histone H3.3 antibody [EPR17899] - ChIP Grade (ab176840) at 1/1000 dilution

    Lane 1 : HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysate
    Lane 2 : NIH/3T3 (Mouse embyro fibroblast cells) whole cell lysate

    Lysates/proteins at 10 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

    Predicted band size: 15 kDa
    Observed band size: 15 kDa


    Exposure time: 3 minutes


    Blocking/Dilution buffer: 5% NFDM/TBST.

  • Dot blot analysis of Histone H3.3 peptide (Lane 1), and Histone H3.1 peptide (Lane 2), labeled using ab176840 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated secondary antibody at 1/1000 dilution.

    Blocking/Dilution buffer: 5% NFDM/TBST.

  • All lanes : Anti-Histone H3.3 antibody [EPR17899] - ChIP Grade (ab176840) at 1/1000 dilution

    Lane 1 : HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysate
    Lane 2 : HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysate with Histone H3.3 peptide
    Lane 3 : HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysate with Histone H3.1 peptide
    Lane 4 : NIH/3T3 (Mouse embyro fibroblast cells) whole cell lysate
    Lane 5 : NIH/3T3 (Mouse embyro fibroblast cells) whole cell lysate with Histone H3.3 peptide
    Lane 6 : NIH/3T3 (Mouse embyro fibroblast cells) whole cell lysate with Histone H3.1 peptide

    Lysates/proteins at 10 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

    Predicted band size: 15 kDa
    Observed band size: 15 kDa


    Exposure time: 3 minutes


    Blocking experiment by pre-incubating Histone H3.3 peptide with the antibody in lanes 2 and 5 and Histone H3.1 peptide in lanes 3 and 6 to show the antibody specificity against Histone H3.3.

    Blocking/Dilution buffer: 5% NFDM/TBST.

  • Chromatin was prepared from HeLa (Human epithelial cells from cervix adenocarcinoma) 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 ab176840 (blue), and 20µl of Anti rabbit IgG sepharose beads. 2μg of rabbit normal IgG was added to the beads control (yellow). The immunoprecipitated DNA was quantified by real time PCR (Sybr green approach).

  • Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (Human epithelial cells from cervix adenocarcinoma) cells labeling Histone H3.3 with ab176840 at 1/1000 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/500 dilution (green). Confocal image showing nuclear staining on HeLa cell line. The nuclear counter stain is DAPI (blue). Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution and ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution (red).

    The negative controls are as follows:
    -ve control 1: ab176840 at 1/1000 dilution followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution.
    -ve control 2: ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution followed by ab150077 (Alexa Fluor®488 Goat Anti-Rabbit IgG H&L) at 1/500 dilution.

  • Immunohistochemical analysis of paraffin-embedded Human colon tissue labeling Histone H3.3 with ab176840 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody at 1/500 dilution. Nucleus staining on Human colon tissue is observed. Counter stained with Hematoxylin.

    Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

  • Immunohistochemical analysis of paraffin-embedded Mouse stomach tissue labeling Histone H3.3 with ab176840 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody at 1/500 dilution. Nucleus staining on mouse stomach tissue is observed. Counter stained with Hematoxylin.

    Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

  • Immunohistochemical analysis of paraffin-embedded rat colon tissue labeling Histone H3.3 with ab176840 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody at 1/500 dilution. Nucleus staining on Rat colon tissue is observed. Counter stained with Hematoxylin.

    Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

References

This product has been referenced in:
  • Tvardovskiy A  et al. Accumulation of histone variant H3.3 with age is associated with profound changes in the histone methylation landscape. Nucleic Acids Res 45:9272-9289 (2017). Read more (PubMed: 28934504) »
See 1 Publication for this product

Customer reviews and Q&As

Application
Western blot
Sample
Mouse Cell lysate - whole cell (mouse embryonic stem cells (J1))
Gel Running Conditions
Reduced Denaturing
Loading amount
20 µg
Specification
mouse embryonic stem cells (J1)
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 22°C

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Submitted Nov 02 2018

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