Recombinant
RabMAb

Anti-Histone H3 antibody [EPR16987] - Nuclear Loading Control and ChIP Grade (ab176842)

Overview

  • Product name
    Anti-Histone H3 antibody [EPR16987] - Nuclear Loading Control and ChIP Grade
    See all Histone H3 primary antibodies
  • Description
    Rabbit monoclonal [EPR16987] to Histone H3 - Nuclear Loading Control and ChIP Grade
  • Tested applications
    Suitable for: IHC-P, ICC/IF, PepArr, WB, ChIP, Flow Cytmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Human, Saccharomyces cerevisiae, Drosophila melanogaster, Schizosaccharomyces pombe
    Predicted to work with: a wide range of other species
  • Immunogen

    Synthetic peptide (the amino acid sequence is considered to be commercially sensitive) within Human Histone H3 aa 100 to the C-terminus. The exact sequence is proprietary.
    Database link: P68431

  • Positive control
    • WB: HeLa, NIH3T3, Saccharomyces cerevisiae and Schizosaccharomyces pombe whole cell lysates. Drosophila embryo nuclear extract lysate. IHC-P: Human colon, Mouse liver, and Rat pancreas tissues. ICC/IF: HeLa cells. ChIP: HeLa chromatin.
  • 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

Applications

Our Abpromise guarantee covers the use of ab176842 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
IHC-P 1/5000 - 1/10000. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
ICC/IF 1/2000 - 1/5000.
PepArr Use at an assay dependent concentration.
WB Use a concentration of 1 µg/ml. Detects a band of approximately 17 kDa (predicted molecular weight: 15 kDa).
ChIP Use 2 µg for 25 µg of chromatin.
Flow Cyt Use at an assay dependent concentration.

Target

  • Function
    Core 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 similarities
    Belongs to the histone H3 family.
  • Developmental stage
    Expressed 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 localization
    Nucleus. 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

Images

  • Chromatin was prepared from HeLa 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 ab176842 (blue), and 20µl of Protein A/G sepharose beads. No antibody was added to the beads control (yellow). The immunoprecipitated DNA was quantified by real time PCR (Taqman approach for active and inactive loci, Sybr green approach for heterochromatic loci). Primers and probes are located in the first kb of the transcribed region.

  • Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (Human epithelial cells from cervix adenocarcinoma) cells labeling Histone H3 with ab176842 at 1/2000 dilution, followed by Goat anti-rabbit Alexa Fluor® 488 (IgG) (ab150077) secondary antibody at 1/400 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 1/500 dilution and ab150120 (goat anti-mouse AlexaFluor®594 Goat secondary antibody) at 1/500 dilution (red).
    The negative controls are as follows;
    -ve control 1: ab176842 at 1/2000 dilution followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution.
    -ve control 2: ab7291 (anti-Tubulin mouse mAb) at 1/500 dilution followed by ab150077 (Alexa Fluor®488 Goat Anti-Rabbit IgG H&L) at 1/400 dilution.

     

  • All lanes : Anti-Histone H3 antibody [EPR16987] - Nuclear Loading Control and ChIP Grade (ab176842) at 1 µg

    Lane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate at 10 µg
    Lane 2 : NIH 3T3 (Mouse embryonic fibroblast cell line) Whole Cell Lysate at 10 µg
    Lane 3 : Drosophila embryo nuclear extract (from melanogaster embryos 0-12hr) at 10 µg
    Lane 4 : S.cerevisiae (Y190) Whole Cell Lysate at 20 µg
    Lane 5 : S.pombe Whole Cell Lysate at 20 µg

    Secondary
    Peroxidase AffiniPure Goat Anti-Rabbit IgG (H+L) at 1/50000 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 : 5 seconds

    This blot was produced using a 4-12% Bis-tris gel under the MES buffer system. The gel was run at 200V for 35 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 2% Bovine Serum Albumin before being incubated with ab176842 overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP, and visualised using ECL development solution ab133406.

  • Immunohistochemical analysis of paraffin-embedded Human colon tissue labeling Histone H3 with ab176842 at 1/500 dilution, followed by Goat Anti-Rabbit HRP (IgG; H&L) secondary antibody (ab97051) at 1/500 dilution. Nucleus staining on glandular epithelium of Human colon tissue is observed. Counter stained with Hematoxylin.

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

  • Immunohistochemical analysis of paraffin-embedded Mouse liver tissue labeling Histone H3 with ab176842 at 1/500 dilution, followed by Goat Anti-Rabbit HRP (IgG H&L) secondary antibody (ab97051) at 1/500 dilution. Nucleus staining on mouse liver tissue is observed. Counter stained with Hematoxylin.

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

  • Immunohistochemical analysis of paraffin-embedded Rat pancreas tissue labeling Histone H3 with ab176842 at 1/500 dilution, followed by Goat Anti-Rabbit HRP (IgG H&L) secondary antibody (ab97051) at 1/500 dilution. Nucleus staining on rat pancreas tissue is observed. Counter stained with Hematoxylin.

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

  • Flow Cytometry analysis of HeLa (human cervix adenocarcinoma) cells labeling Histone H3 with purified ab176842 at 1/40 dilution (10ug/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) was used as the unlabeled control.

  • All batches of ab176842 are tested in Peptide Array against 501 different modified and unmodified histone peptides; each peptide is printed on the array at six concentrations (each in triplicate).
    Circle area represents affinity between the antibody and a peptide: all antigen-containing peptides are displayed as red circles, all other peptides as blue circles. The affinity is calculated as area under curve when antibody binding values are plotted against the corresponding peptide concentration. Each circle area is normalized to the peptide with the strongest affinity.
    The complete dataset, including full list of all peptides and information on the position of each peptide in the diagram, can be downloaded here.

References

This product has been referenced in:
  • Baehr CA  et al. Glycogen Synthase Kinase 3 (GSK-3)-mediated Phosphorylation of Uracil N-Glycosylase 2 (UNG2) Facilitates the Repair of Floxuridine-induced DNA Lesions and Promotes Cell Survival. J Biol Chem 291:26875-26885 (2016). Read more (PubMed: 27875297) »

See 1 Publication for this product

Customer reviews and Q&As

Application
Immunoprecipitation
Sample
Human Cell lysate - nuclear (MDA-MB-231 cells)
Total protein in input
1e+007 cells
Immuno-precipitation step
Other - Protein G Dynabeads
Specification
MDA-MB-231 cells
Username

Ifeoluwa Adewumi

Verified customer

Submitted Feb 28 2017

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Human Cell lysate - whole cell (MCF 7 CELLS)
Gel Running Conditions
Reduced Denaturing (15%)
Loading amount
10 µg
Treatment
20ng/ml TNF alpha
Specification
MCF 7 CELLS
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 22°C
Username

Ifeoluwa Adewumi

Verified customer

Submitted Feb 14 2017

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (MCF7)
Permeabilization
Yes - see blocking buffer
Specification
MCF7
Blocking step
Serum as blocking agent for 30 minute(s) · Concentration: 1% · Temperature: rt°C
Fixative
Methanol
Username

Abcam user community

Verified customer

Submitted Apr 06 2016

Application
Western blot
Sample
Human Recombinant protein (Recombinant octamer & purified K562 cell histone)
Gel Running Conditions
Reduced Denaturing (18%)
Loading amount
0.5 µg
Specification
Recombinant octamer & purified K562 cell histone
Blocking step
Odyssey blocking solution 1/1 (vol/vol) PBS as blocking agent for 16 hour(s) and 0 minute(s) · Concentration: 50% · Temperature: 4°C
Username

Dr. Ragnhild Eskeland

Verified customer

Submitted May 11 2015

Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Human Tissue sections (Colon)
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: Citric acid
Permeabilization
No
Specification
Colon
Blocking step
BSA as blocking agent for 10 minute(s) · Concentration: 1% · Temperature: 21°C
Fixative
Formaldehyde
Username

Mr. Carl Hobbs

Verified customer

Submitted Apr 20 2015

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

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