Overview

  • Product name
    Anti-Histone H3.3 antibody
    See all Histone H3.3 primary antibodies
  • Description
    Rabbit polyclonal to Histone H3.3
  • Host species
    Rabbit
  • Tested applications
    Suitable for: ELISA, ICC/IF, IHC-P, WB, ChIPmore details
  • Species reactivity
    Reacts with: Rat, Human
  • Immunogen

    Synthetic peptide corresponding to Human Histone H3.3. (synthesized peptide derived from Internal of human Histone H3.3.)
    Database link: P84243

  • Positive control
    • Hela cells and human brain tissue

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. Avoid freeze / thaw cycle.
  • Storage buffer
    pH: 7.40
    Preservative: 0.02% Sodium azide
    Constituents: PBS, 50% Glycerol, 0.87% Sodium chloride
  • Concentration information loading...
  • Purity
    Immunogen affinity purified
  • Purification notes
    The antibody was affinity purified from rabbit antiserum by affinity chromatography using epitope specific immunogen.
  • Clonality
    Polyclonal
  • Isotype
    IgG
  • Research areas

Applications

Our Abpromise guarantee covers the use of ab62642 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
ELISA 1/5000.
ICC/IF 1/500 - 1/1000.
IHC-P 1/50 - 1/100.
WB Use a concentration of 1 µg/ml. Detects a band of approximately 19 kDa (predicted molecular weight: 15 kDa).
ChIP Use at an assay dependent concentration.

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 of Histone H3.3 from primary adipose chromatin using ab62642, showing enrichment at promoter regions previously associated with Histone H3.3 enrichment. The ChIP experiment was performed according to Dahl and Collas, 2007 (please see accompanying abreview for further details).

    See Abreview

  • Immunofluorescence analysis of HeLa cells, using ab62642 at a 1/500 dilution.
    Left image untreated.
    Right image treated with peptide.
  • Immunohistochemistry analysis of paraffin embedded human brain tissue using ab62642 at a 1/50 dilution.
    Left image untreated.
    Right image treated with peptide.
  • Anti-Histone H3.3 antibody (ab62642) at 1 µg/ml + A431 (Human epithelial carcinoma cell line) Whole Cell Lysate at 10 µg

    Secondary
    Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (ab97080) at 1/5000 dilution

    Developed using the ECL technique.

    Performed under reducing conditions.

    Predicted band size: 15 kDa
    Observed band size: 19 kDa (why is the actual band size different from the predicted?)
    Additional bands at: 45 kDa. We are unsure as to the identity of these extra bands.


    Exposure time: 12 minutes


    The 19 kDa band observed is comparable to the molecular weight seen with other commercially available antibodies to Histone H3.3.

References

This product has been referenced in:
  • Rachdaoui N  et al. Turnover of histones and histone variants in postnatal rat brain: effects of alcohol exposure. Clin Epigenetics 9:117 (2017). Read more (PubMed: 29075360) »
  • Veith N  et al. Mechanisms underlying epigenetic and transcriptional heterogeneity in Chinese hamster ovary (CHO) cell lines. BMC Biotechnol 16:6 (2016). ChIP . Read more (PubMed: 26800878) »

See all 19 Publications for this product

Customer reviews and Q&As

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Human Cell lysate - whole cell (HEK293T cells cultured in DMEME+10% FBS)
Gel Running Conditions
Reduced Denaturing (15% precast Bis-Tris gel)
Loading amount
20 µg
Treatment
H3.3 with V5 tag over-expressed in pTRIEX 3 plasmid. Harvested 48h post-transfection
Specification
HEK293T cells cultured in DMEME+10% FBS
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
Username

Abcam user community

Verified customer

Submitted Dec 19 2017

Abcam has not validated the combination of species/application used in this Abreview.
Application
Western blot
Sample
Zebrafish Cell lysate - whole cell (whole embryo, deyolked)
Gel Running Conditions
Reduced Denaturing (NuPAGE Bis-Tris gel 4-12%)
Loading amount
20000 cells
Specification
whole embryo, deyolked
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
Username

Abcam user community

Verified customer

Submitted Jun 24 2013

Abcam has not validated the combination of species/application used in this Abreview.
Application
Western blot
Sample
Mouse Cell lysate - whole cell (Raw 264.7)
Gel Running Conditions
Reduced Denaturing (12)
Loading amount
25 µg
Treatment
DRB and LPS 0-2hrs
Specification
Raw 264.7
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 23°C
Username

Abcam user community

Verified customer

Submitted Jul 31 2012

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (osteosarcoma cell line, Saos2)
Permeabilization
Yes - PBS TRITON .25%
Specification
osteosarcoma cell line, Saos2
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: RT°C
Fixative
Paraformaldehyde
Username

Abcam user community

Verified customer

Submitted Apr 18 2012

To our knowledge, ab62642 has not been tested in bull. Therefore, I can offer a discount off a future purchase if you buy ab62642 now, test it inbull and submit feedback to us in the form of an Abreview. It doesn't matter whether the Abreview is positi...

Read More
Abcam guarantees this product to work in the species/application used in this Abreview.
Application
ChIP
Sample
Human Cell lysate - nuclear (Colon cancer cell)
Negative control
IgG control
Specification
Colon cancer cell
Detection step
Real-time PCR
Type
Cross-linking (X-ChIP)
Duration of cross-linking step: 10 minute(s) and 0 second(s)
Username

Dr. santosh chauhan

Verified customer

Submitted Aug 02 2011

Abcam has not validated the combination of species/application used in this Abreview.
Application
Immunocytochemistry/ Immunofluorescence
Sample
Zebra finch Cell (HEK293 transfected with zebra finch H3.3 (GFP))
Permeabilization
Yes - 0.3% TritonX-100
Specification
HEK293 transfected with zebra finch H3.3 (GFP)
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: 25°C
Fixative
Paraformaldehyde
Username

Abcam user community

Verified customer

Submitted May 27 2010

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
ChIP
Sample
Human Cell lysate - whole cell (Soluble chromatin preparation from primary adipose)
Negative control
No antibody Dynabeads Protein A-only control
Specification
Soluble chromatin preparation from primary adipose
Detection step
Real-time PCR
Type
Cross-linking (X-ChIP)
Duration of cross-linking step: 8 minute(s) and 0 second(s)
Specification of the cross-linking agent: Formaldehyde, 1%
Positive control
Promoter regions for which we have strong evidence of epitope tagged H3.3 enrichment in the human genome data provided byh ChIP-on-chip and ChIP-qPCR.
Username

Prof. Philippe Collas

Verified customer

Submitted Oct 01 2009

Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"

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