Overview

  • Product nameAnti-Histone H3 antibody
    See all Histone H3 primary antibodies
  • Description
    Rabbit polyclonal to Histone H3
  • Specificityab18521 recognises unmodified and modified forms of histone H3
  • Tested applicationsSuitable for: IP, WB, IHC-Pmore details
  • Species reactivity
    Reacts with: Cow, Human, Saccharomyces cerevisiae, Xenopus laevis
    Predicted to work with: Mouse, Arabidopsis thaliana, Caenorhabditis elegans, Drosophila melanogaster
  • Immunogen

    Synthetic peptide corresponding to Human Histone H3 aa 1-100 (N terminal) conjugated to Keyhole Limpet Haemocyanin (KLH).
    Database link: P68431
    (Peptide available as ab14949)

  • Positive control
    • This antibody gave a positive signal in Calf Thymus Histone Lysate - Nuclear Prep, Colcemid Treated Hela Histone Prep, Histone H3.1 Recombinant. IHC-P: human testis tissue sections.

Properties

Applications

Our Abpromise guarantee covers the use of ab18521 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 Use a concentration of 5 µg/ml.
WB Use a concentration of 1 µg/ml. Detects a band of approximately 17 kDa (predicted molecular weight: 15 kDa).
IHC-P Use a concentration of 1 µg/ml. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

Target

  • FunctionCore 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 similaritiesBelongs to the histone H3 family.
  • Developmental stageExpressed 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 localizationNucleus. Chromosome.
  • Information by UniProt
  • Database links
  • Alternative names
    • H3 histone family, member A antibody
    • H3/A antibody
    • H31_HUMAN antibody
    • H3FA antibody
    • Hist1h3a antibody
    • HIST1H3B antibody
    • HIST1H3C antibody
    • HIST1H3D antibody
    • HIST1H3E antibody
    • HIST1H3F antibody
    • HIST1H3G antibody
    • HIST1H3H antibody
    • HIST1H3I antibody
    • HIST1H3J antibody
    • histone 1, H3a antibody
    • Histone cluster 1, H3a 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

Anti-Histone H3 antibody images

  • Histone H3 was immunoprecipitated using 0.5mg Hela whole cell extract, 5µg of Rabbit polyclonal to Histone H3 and 50µl of protein G magnetic beads (+). No antibody was added to the control (-).

    The antibody was incubated under agitation with Protein G beads for 10min, Hela whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.

    Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70°C; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab18521.

    Secondary: Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697).

    Band: 17kDa; Histone H3

  • All lanes : Anti-Histone H3 antibody (ab18521) at 1 µg/ml

    Lane 1 : Calf Thymus Histone Preparation Nuclear Lysate at 0.5 µg
    Lane 2 : HeLa Histone Preparation Nuclear Lysate - Colcemid-treated at 2.5 µg
    Lane 3 : Histone H2A Recombinant Protein at 0.1 µg
    Lane 4 : Histone H3.1 Recombinant Protein at 0.1 µg
    Lane 5 : Histone H4 Recombinant Protein at 0.1 µg

    Secondary
    Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

    Performed under reducing conditions.

    Predicted band size : 15 kDa
    Observed band size : 17 kDa (why is the actual band size different from the predicted?)
  • ab18521 staining Histone H3 in human MEF cells by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with paraformaldehyde, permeabilized with Triton X-100 and blocked with 1% serum for 10 minutes at 21°C. Samples were incubated with primary antibody (1/3000) for 10 hours at 21°C. A FITC-conjugated goat anti-rabbit IgG polyclonal (1/500) was used as the secondary antibody.

    See Abreview

  • ICC/IF image of ab18521 stained Hek293 cells. The cells were 100% methanol fixed (5 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab18521, 1æg/ml) overnight at +4øC. The secondary antibody (green)ÿwas Alexa Fluor© 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor© 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43æM.

  • All lanes : Anti-Histone H3 antibody (ab18521) at 1/250 dilution

    Lane 1 : Whole cell lysate prepared from Saccharomyces cerevisiae WT (BY4741).
    Lane 2 : Whole cell lysate prepared from Saccharomyces cerevisiae delta hht1 HHT2.
    Lane 3 : Whole cell lysate prepared from Saccharomyces cerevisiae delta hht1 hh2-K9, 14, 18R.

    Lysates/proteins at 500000 cells per lane.

    Secondary
    HRP conjugated goat anti-rabbit IgG at 1/10000 dilution
    Developed using the ECL technique

    Performed under reducing conditions.

    Predicted band size : 15 kDa
    Observed band size : 17 kDa (why is the actual band size different from the predicted?)


    Exposure time : 10 minutes

    This image was kindly supplied by Dr Tobias Eisenberg by Abreview

    Blocking performed using 3% BSA for 16 hours at 4°C.
    Lysates prepared using 5% Trichloroethane.

    See Abreview

  • IHC image of Histone H3 staining in human testis formalin fixed paraffin embedded tissue section, performed on a Leica BondTM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab18521, 1µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

    For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.
  • All lanes : Anti-Histone H3 antibody (ab31602) at 1 µg/ml

    Lane 1 : Calf thymus histone lysate
    Lane 2 : Calf thymus histone lysate with Human Histone H3 peptide (ab14949) at 1 µg/ml
    Lane 3 : Calf thymus histone lysate with Human Histone H3 (mono methyl K4) peptide (ab1340) at 1 µg/ml
    Lane 4 : Calf thymus histone lysate with Human Histone H3 (di methyl K4) peptide (ab7768) at 1 µg/ml
    Lane 5 : Calf thymus histone lysate with Human Histone H3 (tri methyl K4) peptide (ab1342) at 1 µg/ml

    Lysates/proteins at 20 µg per lane.


    Predicted band size : 15 kDa
    Observed band size : 15 kDa
    ab18521 recognises histone H3 as well as modified histone H3 at approximately 15 kDa(lane1). This is demonstrated by the blocking using the immunising unmodified histone H3 peptide (lane2), histone H3 mono methyl K4 (lane3), histone H3 di methyl K4 (lane4) and histone H3 tri methyl K4 (lane5).

References for Anti-Histone H3 antibody (ab18521)

This product has been referenced in:
  • Canovas S  et al. Jumonji domain-containing protein 3 regulates histone 3 lysine 27 methylation during bovine preimplantation development. Proc Natl Acad Sci U S A 109:2400-5 (2012). Read more (PubMed: 22308433) »
  • Hyun S  et al. An RNA aptamer that selectively recognizes symmetric dimethylation of arginine 8 in the histone h3?n-terminal Peptide. Nucleic Acid Ther 21:157-63 (2011). Read more (PubMed: 21749292) »

See all 4 Publications for this product

Product Wall

Abcam guarantees this product to work in the species/application used in this Abreview.
Application Western blot
Sample Human Cell lysate - nuclear (Lymphocytes)
Gel Running Conditions Reduced Denaturing (4-12% Bis-Tris)
Loading amount 30 µg
Specification Lymphocytes
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 Sep 05 2016

Application Immunocytochemistry/ Immunofluorescence
Blocking step Serum as blocking agent for 10 minute(s) · Concentration: 1% · Temperature: 21°C
Sample Human Cell (MEF)
Specification MEF
Permeabilization Yes - TRITON
Fixative Paraformaldehyde
Username

Abcam user community

Verified customer

Submitted Apr 24 2014

Application Western blot
Loading amount 200000 cells
Gel Running Conditions Reduced Denaturing (12 %, SDS GEL)
Sample Human Cell lysate - whole cell (HEK293)
Specification HEK293
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 21°C
Username

Dr. Edyta Marcon

Verified customer

Submitted Apr 18 2014

Abcam guarantees this product to work in the species/application used in this Abreview.
Application Western blot
Sample Xenopus laevis Cell lysate - whole cell (Erythrocytes)
Loading amount 50000 cells
Specification Erythrocytes
Gel Running Conditions Reduced Denaturing (12%)
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 22°C
Username

Abcam user community

Verified customer

Submitted Apr 01 2013

Abcam guarantees this product to work in the species/application used in this Abreview.
Application Western blot
Sample Saccharomyces cerevisiae Cell lysate - whole cell (5% TCA lysate preparation with glass beads accordi)
Loading amount 500000 cells
Specification 5% TCA lysate preparation with glass beads accordi
Treatment stationary cells
Gel Running Conditions Reduced Denaturing (15%)
Blocking step BSA as blocking agent for 16 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: 4°C
Username

Herr Dr. Tobias Eisenberg

Verified customer

Submitted Mar 29 2010

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