Recombinant
RabMAb

Recombinant Anti-Histone H3.3 (phospho S31) antibody [EPR1873] (ab92628)

Overview

  • Product name

    Anti-Histone H3.3 (phospho S31) antibody [EPR1873]
    See all Histone H3.3 primary antibodies
  • Description

    Rabbit monoclonal [EPR1873] to Histone H3.3 (phospho S31)
  • Host species

    Rabbit
  • Specificity

    Detects Histone H3.3 when phosphorylated at S31.
  • Tested applications

    Suitable for: Flow Cyt, Dot blot, ChIP, WB, IHC-P, ICC/IFmore details
    Unsuitable for: IP
  • Species reactivity

    Reacts with: Mouse, Rat, Human
  • Immunogen

    Synthetic peptide within Human Histone H3.3 aa 1-100 (phospho S31). The exact sequence is proprietary.
    Database link: P84243

  • Positive control

    • Untreated Hela cells and HeLa cell lysate treated with Calyculin A and/or nocodazole. Human cervical carcinoma
  • 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.

    We are constantly working hard to ensure we provide our customers with best in class antibodies. As a result of this work we are pleased to now offer this antibody in purified format. We are in the process of updating our datasheets. The purified format is designated 'PUR' on our product labels. If you have any questions regarding this update, please contact our Scientific Support team.

    This product is a recombinant rabbit monoclonal antibody.

Properties

  • Form

    Liquid
  • Storage instructions

    Shipped at 4°C. Store at -20°C. Stable for 12 months at -20°C.
  • Storage buffer

    pH: 7.20
    Preservative: 0.01% Sodium azide
    Constituents: 59% PBS, 40% Glycerol, 0.5% BSA
  • Concentration information loading...
  • Purity

    Protein A purified
  • Clonality

    Monoclonal
  • Clone number

    EPR1873
  • Isotype

    IgG
  • Research areas

Applications

Our Abpromise guarantee covers the use of ab92628 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
Flow Cyt Use at an assay dependent concentration.
Dot blot 1/1000.
ChIP Use at an assay dependent concentration. PubMed: 22649058
WB 1/1000 - 1/5000. Predicted molecular weight: 15 kDa.
IHC-P 1/100 - 1/250.
ICC/IF 1/100 - 1/250.
  • Application notes
    Is unsuitable for IP.
  • 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 (phospho S31) antibody [EPR1873] (ab92628) at 1/1000 dilution

      Lane 1 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate
      Lane 2 : Whole cell lysate from HeLa (Human epithelial cell line from cervix adenocarcinoma) cells treated with 100ng/ml of nocodazole for 18 hours
      Lane 3 : Whole cell lysate from HeLa (Human epithelial cell line from cervix adenocarcinoma) cells treated with 100ng/ml of nocodazole for 18 hours. Membrane incubated with phosphatase

      Lysates/proteins at 15 µg per lane.

      Secondary
      All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

      Predicted band size: 15 kDa


      Exposure time: 15 seconds


      Blocking/dilution buffer: 2% BSA/TBST

    • Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% tritonX-100 HeLa (Human epithelial cell line from cervix adenocarcinoma) cells, LP starved and non-starved, labeling anti-Histone H3.3 (phospho S31) with Ab92628 at 1/500 dilution followed by Goat anti-Rabbit secondary IgG AlexaFluor®488 (ab150077) secondary antibody at 1/1000 dilution (green).

      Confocal image showing nuclear staining on M phase of HeLa cells, then the signal decreased after LP treatment.

      For the pan antibody, there was no great difference after LP treatment. The data showed mostly nuclear staining.

    • Paraffin-embedded human cervical carcinoma labelled with ab92628 at 1/100 dilution.

      Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

    • Flow Cytometry analysis of Hela (human cervix adenocarcinoma) cells treated with 100 ng/ml Nocodazole for 18 hours, labeling Histone H3.3 with purified ab92628 at 1/150 dilution (10µg/ml) (red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. A Goat anti-rabbit IgG (Alexa Fluor® 488) (1/2000 dilution) was used as the secondary antibody. Rabbit monoclonal IgG (Black) was used as the isotype control, cells without incubation with primary antibody and secondary antibody (Blue) were used as the unlabeled control. Untreated control - Hela (human cervix adenocarcinoma) cells untreated with 100 ng/ml Nocodazole for 18 hours (Green).

    • Dot Blot analysis of Lane 1: Histone H3.3 (pS31) phospho peptide and Lane 2: Histone H3.3 non-phospho peptide labeling Histone H3.3 (phospho S31) with ab92628 at 1/1000 dilution. 5% NFDM/TBST was used as the diluting and blocking buffer. 

      ab97051 Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated was used as the secondary antibody at 1/100000 dilution. Exposure time: 10 seconds.

       

       

       

       

       

    • All lanes : Anti-Histone H3.3 (phospho S31) antibody [EPR1873] (ab92628) at 1/1000 dilution

      Lane 1 : HeLa cell lysate untreated
      Lane 2 : HeLa cell lysate treated with Calyculin A

      Lysates/proteins at 10 µg per lane.

      Secondary
      All lanes : HRP labelled goat anti-rabbit at 1/2000 dilution

      Predicted band size: 15 kDa

    • Representative western blot detecting Histone H3.3 (phospho S31) using ab92628 at 1/3000 dilution.

      Recombinant Histone H3.3 protein was treated with recombinant kinase and 200 µM ATP for the indicated times. 0.2 µg of protein was loaded to each lane and phosphorylated Histone H3.3 was detected using ab92628. An HRP-conjugated goat anti-rabbit polyclonal (1/20000) was used as the secondary antibody

      See Abreview

    References

    This product has been referenced in:

    • Tschuor C  et al. Yes-associated protein promotes early hepatocyte cell cycle progression in regenerating liver after tissue loss. FASEB Bioadv 1:51-61 (2019). Read more (PubMed: 30740593) »
    • Leidecker O  et al. Serine is a new target residue for endogenous ADP-ribosylation on histones. Nat Chem Biol 12:998-1000 (2016). Read more (PubMed: 27723750) »
    See all 7 Publications for this product

    Customer reviews and Q&As

    1-4 of 4 Abreviews or Q&A

    Application
    Western blot
    Sample
    Chicken Recombinant protein (DT40)
    Gel Running Conditions
    Reduced Denaturing (4-12)
    Loading amount
    500000 cells
    Specification
    DT40
    Blocking step
    BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C

    Ms. Marina Romanello

    Verified customer

    Submitted Jun 02 2015

    Application
    Immunocytochemistry/ Immunofluorescence
    Sample
    Mouse Cell (Testis)
    Permeabilization
    Yes - Triton X100
    Specification
    Testis
    Blocking step
    BSA as blocking agent for 30 minute(s) · Concentration: 10% · Temperature: 20°C
    Fixative
    Paraformaldehyde

    Abcam user community

    Verified customer

    Submitted Dec 23 2013

    Application
    Western blot
    Sample
    Mouse Cell lysate - whole cell (Raw 264.7)
    Gel Running Conditions
    Reduced Denaturing (10)
    Loading amount
    20 µg
    Treatment
    DRB and Vehicle control 0 - 2hrs
    Specification
    Raw 264.7
    Blocking step
    BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 2% · Temperature: 23°C

    Abcam user community

    Verified customer

    Submitted Jul 31 2012

    Application
    Western blot
    Sample
    Human Recombinant protein (H3.3 recombinant protein)
    Gel Running Conditions
    Reduced Denaturing (10%)
    Loading amount
    0.2 µg
    Treatment
    Kinase and 200uM ATP
    Specification
    H3.3 recombinant protein
    Blocking step
    BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: 22°C

    Abcam user community

    Verified customer

    Submitted Jun 08 2012

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