Anti-Histone H3 antibody - Nuclear Loading Control and ChIP Grade (ab1791)

Overview

  • Product nameAnti-Histone H3 antibody - Nuclear Loading Control and ChIP GradeSee all Histone H3 primary antibodies ...
  • Description
    Rabbit polyclonal to Histone H3 - Nuclear Loading Control and ChIP Grade
  • Tested applicationsIHC-Fr, CHIPseq, Dot Blot, Flow Cyt, IHC-P, Electron Microscopy, ICC/IF, ChIP, IP, WB, ChIP/Chip, IHC - Wholemount, ICC more details
  • Species reactivity
    Reacts with: Mouse, Rat, Chicken, Dog, Human, Saccharomyces cerevisiae, Xenopus laevis, Arabidopsis thaliana, Caenorhabditis elegans, Fruit fly (Drosophila melanogaster), Ferret, Indian Muntjac, Schizosaccharomyces pombe, Zebrafish, Trypanosoma cruzi, Neurospora crassa , Toxoplasma gondii, Rice, Schistosoma mansoni , Candida albicans, Cyanidioschyzon merolae
    Predicted to work with: a wide range of other species, all Mammals
  • Immunogen

    Synthetic peptide conjugated to KLH derived from within residues 100 to the C-terminus of Human Histone H3.

    (Peptide available as ab12149.)

  • Positive control
    • HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate. Available to buy from Abcam under ab150035

Properties

  • FormLiquid
  • Storage instructionsShipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C or -80°C. Avoid freeze / thaw cycle.
  • Storage bufferpH: 7.40
    Preservative: 0.02% Sodium azide
    Constituent: PBS
    Note: Batches of this product that have a concentration < 1mg/ml may have BSA added as a stabilising agent. If you would like information about the formulation of a specific lot, please contact our scientific support team who will be happy to help.
  • Concentration information loading...
  • PurityImmunogen affinity purified
  • Clonality Polyclonal
  • IsotypeIgG
  • Research Areas

Applications

Our Abpromise guarantee covers the use of ab1791 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Notes
IHC-Fr 1/200.
CHIPseq Use at an assay dependent concentration.
Dot Blot 1/10000.
Flow Cyt Use at an assay dependent concentration.
IHC-P 1/100 - 1/400. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
Electron Microscopy 1/50. Customer feedback
ICC/IF Use a concentration of 1 µg/ml.
ChIP Use 2µg for 106 cells.
IP Use a concentration of 5 µg/ml.
WB 1/1000 - 1/5000. Detects a band of approximately 17 kDa (predicted molecular weight: 15 kDa).Can be blocked with Human Histone H3 peptide (ab12149).
ChIP/Chip Use at an assay dependent concentration.
IHC - Wholemount Use at an assay dependent concentration. PubMed: 22219645
ICC 1/100 - 1/500.

Target

  • FunctionVariant 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 similaritiesBelongs to the histone H3 family.
  • Developmental stageExpressed 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 localizationNucleus. Chromosome.
  • Target information above from: UniProt accession P84243 The UniProt Consortium
    The Universal Protein Resource (UniProt) in 2010
    Nucleic Acids Res. 38:D142-D148 (2010) .

    Information by UniProt
  • Database links
    see all
  • Alternative names
    • H3.3B antibody
    • H3F3A antibody
    • H3 histone antibody
    • H3 histone family member E pseudogene antibody
    • H3 histone family member E pseudogene antibody
    • H3.3A antibody
    • H33_HUMAN antibody
    • H3F3 antibody
    • H3F3 antibody
    • H3f3b antibody
    • HIST3H3 antibody
    • HIST3H3 antibody
    • Histone H3 3 pseudogene antibody
    • Histone H3 3 pseudogene antibody
    • Histone H3.3 antibody
    • PP781 antibody
    see all

Anti-Histone H3 antibody - Nuclear Loading Control and ChIP Grade images

  • Histone H3 - ChIP Grade was immunoprecipitated using 0.5mg HeLa whole cell extract, 5µg of Rabbit polyclonal to 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 ab1791.

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

    Band: 15kDa; Histone H3 - ChIP Grade

  • All lanes : Anti-Histone H3 antibody - Nuclear Loading Control and ChIP Grade (ab1791) at 1/1000 dilution

    Lane 1 : A431 Whole Cell Lysate
    Lane 2 : Jurkat Whole Cell Lysate
    Lane 3 : 293 Whole Cell Lysate
    Lane 4 : A431 Whole Cell Lysate with Human Histone H3 peptide (ab12149) at 1 µg/ml
    Lane 5 : Jurkat Whole Cell Lysate with Human Histone H3 peptide (ab12149) at 1 µg/ml
    Lane 6 : 293 Whole Cell Lysate with Human Histone H3 peptide (ab12149) at 1 µg/ml

    Lysates/proteins at 20 µg per lane.

    Secondary
    Goat Anti-Rabbit IgG H&L (HRP) (ab6721) at 1/5000 dilution (Goat polyclonal to Rabbit IgG - H&L (HRP))
    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 seconds
  • Chromatin was prepared from U2OS cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10 min. The ChIP was performed with 25 µg of chromatin, 2 µg of ab1791 (blue), and 20 µl 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). Primers and probes are located in the first kb of the transcribed region.

  • Hela cells cultured on coverslips were fixed with 4% paraformaldehyde and then stained with ab1791 (green) at a working dilution of 1/500. The DNA stained with DAPI is shown in red. (100x magnification).
  • Chromatin from Xenopus laevis oocytes was prepared according to the Abcam X-ChIP protocol. Oocytes were fixed with formaldehyde for 10 min. The ChIP was performed with 25 mg of chromatin, 3 mg of ab7834 (anti-H3, light blue) and 3 µg of ab1791 (anti-H3, dark blue), and 20 ml of Protein A/G sepharose beads. A non-specific antibody was used as a control (yellow). The immunoprecipitated DNA was quantified by real time PCR (Taqman approach).
  •  ab1791 staining mouse embryonic stem cells by flow cytometry (gated on all living cells). The cell colonies were trypsinized and incubated with the antibody 1ug/1.5 x 105cells in a permeabilization buffer. A PE conjugated goat anti-rabbit antibody was used as the secondary.

    See Abreview

  • ab1791 staining rat testes tissue sections by IHC-P.  Sections were formaldehyde fixed and subjected to heat mediated antigen retrieval prior to blocking with 5% serum for 30 minutes at 22°C.  The primary antibody was diluted 1/400 and incubated with the sample for 30 minutes at 22°C.  A HRP-conjugated goat anti-rabbit antibody diluted 1/400 was used as the secondary.

    See Abreview

  • All lanes : Anti-Histone H3 antibody - Nuclear Loading Control and ChIP Grade (ab1791) at 1 µg/ml

    Lane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate
    Lane 2 : NIH 3T3 (Mouse embryonic fibroblast cell line) Whole Cell Lysate (ab7179)
    Lane 3 : Drosophila embryo nuclear extract (from melanogaster embryos 0-12Hr)
    Lane 4 : S.cerevisiae (Y190) Whole Cell Lysate
    Lane 5 : S.pombe Whole Cell Lysate

    Lysates/proteins at 10 µg per lane.

    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?)
    ab1791 is tested in western blot on a range of species.  We recommend loading higher amounts of protein (20-30ug) to increase the signal in yeast lysates
  • ICC/IF image of ab1791 stained HeLa 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 (ab1791, 1µg/ml) overnight at +4°C. The secondary antibody (green) was 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 1.43µM. This antibody also gave a positive result in 5% PFA fixed (5 min) Hek293, HepG2 and MCF7 cells at 1µg/ml, and in 4% PFA fixed (10 min) HeLa and Hek293 cells at 1µg/ml.
  • ab1791 staining Histone H3 in human benign nevi tissue by Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections). Tissue was fixed with formaldehyde and a heat mediated antigen retrieval step was performed. Samples were then blocked with 2.5% normal horse serum for 20 minutes followed by incubation with the primary antibody at a 1/200 dilution for 16 hours at 4°C. An undiluted HRP-conjugated rabbit polyclonal was used as the secondary antibody.

    See Abreview

  • All lanes : Anti-Histone H3 antibody - Nuclear Loading Control and ChIP Grade (ab1791) at 1/10000 dilution

    Lane 1 : HeLa nuclear lysate
    Lane 2 : HeLa nuclear lysate
    Lane 3 : HeLa nuclear lysate

    Lysates/proteins at 2 µg per lane.

    Secondary
    HRP conjugated donkey polyclonal at 1/2000 dilution
    developed using the ECL technique

    Predicted band size : 15 kDa


    Exposure time : 5 seconds

    Image courtesy of Dr Roman Hudec by Abreview.

    See Abreview

  • ICC/IF image of ab1791 stained HeLa cells. The cells were 4% formaldehyde fixed (10 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 (ab1791, 1µg/ml) overnight at +4°C. The secondary antibody (green) was ab96899, DyLight® 488 goat anti-rabbit IgG (H+L) used at a 1/250 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 1.43µM.

  • Histone H3 immunogold detection in HeLa cells.
    Ulltra-thin sections of paraformaldehyde-fixed, Lowicryl K4M embedded cells were incubated sequentially with ab1791 antibody and an anti-rabbit antibody coupled to 10 nm gold particles. Notice absence of chromatin within Interchromatin Granules (IG), a reservoir of spliceosomal snRNPs, and high labeling of patches of condensed chromatin close to the nuclear envelope (arrows). Nu: nucleus, Cyt: cytoplasm.

    Image courtesy of Gerard Pierron, IGR-Villejuif, France.

    Sample : human
    Type : HeLa cells
    Fixative : either paraformaldehyde 4% or 1.6% glutaraldehyde in 0.1M Millonig’s buffer.
    Embedding : in Lowicryl 4KM at -20°C under UV.
    Ultrathin-sections deposited on formvar-coated carbonated EM-grids.
    Blocking step: 5% BSA for 30 seconds at RT.
    Primary antibody: Ab1791 diluted 1/50 in PBS, for 1h at RT.
    Secondary antibody: BBI International anti-rabbit



  • Predicted band size : 15 kDa

    John E. Mueller and J. Ruth German (Mary Bryk lab)

    Rabbit polyclonal to Histone H3 (ab1791) at 1/5000 on S. cerevisiae whole cell lysate (40 ug per lane).

    Protein resolved on 15% SDS-PAGE gel. After transfer to PVDF membrane, blots were blocked in 1X PBS, 0.1% Tween-20, and 5% milk. ab1791 was diluted in 5 ml blocking buffer at 1/5000.  Blots plus primary antibodies were either incubated overnight at 4C or at RT for 2 hr. Blots were washed 6X for 10 min each in PBS with 0.1% Tween-20 before addition of secondary antibodies. Secondary antibodies were diluted 1/2,000 in blocking buffer and incubated with blots for 2 hr at RT. Secondary blots were washed 4X for 10 min each in PBS with 0.1% Tween-20 and 2X for 10 min each in PBS.

References for Anti-Histone H3 antibody - Nuclear Loading Control and ChIP Grade (ab1791)

This product has been referenced in:
  • Ueshima S  et al. Upstream binding factor-dependent and pre-rRNA transcription-independent association of pre-rRNA processing factors with rRNA gene. Biochem Biophys Res Commun 443:22-7 (2014). Read more (PubMed: 24269811) »
  • Dawson MA  et al. Recurrent mutations, including NPM1c, activate a BRD4-dependent core transcriptional program in acute myeloid leukemia. Leukemia 28:311-20 (2014). Read more (PubMed: 24220271) »

See all 579 Publications for this product

Product Wall

Thank you for contacting Abcam.

Our antibody ab1791 will recognize total Histone H3 in your samples, so it will recognize the modified forms of Histone H3. Unfortunately I am unaware of the ratio of expression between the different variant for...

Read More

DISCOUNT CODE: XXXXXXX
Expiration date: 17-05-2013
Value: €420

I am very pleased to hear you would like to accept our offer and test ab1791 in Paramecium. This code will give you €420 off your next order before the expiration...

Read More
Application Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Blocking step Heat-Inactivated Normal Donkey Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
Antigen retrieval step None
Sample Mouse Tissue sections (Whole brain sections)
Specification Whole brain sections
Permeabilization Yes - Tween-20
Fixative Paraformaldehyde
Username

Abcam user community

Verified customer

Submitted Apr 11 2014

Application Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Blocking step BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5%
Antigen retrieval step Heat mediated - Buffer/Enzyme Used: Na-citrate pH6
Sample Mouse Tissue sections (Liver)
Specification Liver
Permeabilization Yes - - PBS-Triton 0,05% 30min
Fixative Paraformaldehyde
Username

Abcam user community

Verified customer

Submitted Apr 07 2014

Application Western blot
Loading amount 1 µg
Gel Running Conditions Reduced Denaturing (18% SDS-PAGE)
Sample Dictyostelium discoideum Purified protein (Vegetative cells)
Specification Vegetative cells
Blocking step Using methanol to block PVDF membrane. Soak for 1 minutes and then air dry for 30 minutes. as blocking agent for 30 minute(s) · Concentration: 5% · Temperature: 25°C
Username

Dr. Duen-Wei Hsu

Verified customer

Submitted Mar 31 2014

Application Western blot
Loading amount 25 µg
Gel Running Conditions Non-reduced Denaturing
Sample Mouse Cell lysate - nuclear (primary hepatoctyes)
Specification primary hepatoctyes
Blocking step (agent) for 1 hour(s) and 0 minute(s) · Concentration: 2µg/mL · Temperature: 22°C
Username

Abcam user community

Verified customer

Submitted Mar 05 2014

Application Western blot
Loading amount 30 µg
Gel Running Conditions Reduced Denaturing (15%)
Sample Human Cell lysate - whole cell (SH-SY5Y cell extracts)
Specification SH-SY5Y cell extracts
Treatment with EGF
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 22°C
Username

Dr. Arnaud BERTHIER

Verified customer

Submitted Mar 04 2014

Application Western blot
Loading amount 20 µg
Gel Running Conditions Reduced Denaturing (15%)
Sample Human Cell lysate - whole cell (U2OS osteosarcoma)
Specification U2OS osteosarcoma
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 20°C
Username

Abcam user community

Verified customer

Submitted Feb 21 2014

Application Flow Cytometry
Fixation Paraformaldehyde
Permeabilization Yes - eBioscience Permeabilization Buffer
Sample Human Cell (Differentiated Haematopoietic Stem Cells)
Specification Differentiated Haematopoietic Stem Cells
Gating Strategy Isotype negative control (white)
Preparation Cell harvesting/tissue preparation method: Typsin-EDTA Cell Dissociation
Sample buffer: PBS and 10% FBS
Username

Abcam user community

Verified customer

Submitted Feb 20 2014

Application CHIPseq
Sample Mouse Cell lysate - nuclear (MEF)
Specification MEF
Username

Abcam user community

Verified customer

Submitted Jan 30 2014

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