Overview

  • Product name
    Anti-Histone H4 (acetyl K8) antibody - ChIP Grade
    See all Histone H4 primary antibodies
  • Description
    Rabbit polyclonal to Histone H4 (acetyl K8) - ChIP Grade
  • Host species
    Rabbit
  • Tested applications
    Suitable for: ICC/IF, IHC-P, Electron Microscopy, ChIP, WBmore details
  • Species reactivity
    Reacts with: Mouse, Chicken, Cow, Human, Saccharomyces cerevisiae, Schizosaccharomyces pombe
  • Immunogen

    Synthetic peptide corresponding to Human Histone H4 aa 1-100 (internal sequence) (acetyl K8) conjugated to Keyhole Limpet Haemocyanin (KLH).
    Database link: P62805
    (Peptide available as ab15824)

  • Positive control
    • This antibody gave a positive signal on Hela Histone lysate-Butyrate treated and Calf Thymus Histone lysate.

Properties

Applications

Our Abpromise guarantee covers the use of ab15823 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
ICC/IF Use a concentration of 1 µg/ml.
IHC-P Use a concentration of 1 µg/ml. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
Electron Microscopy Use at an assay dependent concentration. PubMed: 20543957
ChIP Use 2µl for 106 cells.
WB Use a concentration of 1 µg/ml. Detects a band of approximately 12 kDa (predicted molecular weight: 11.2 kDa).

Target

Images

  • 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 ab15823 (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.

  • IHC image of Histone H4 (acetyl K8) staining in human breast carcinoma FFPE section, performed on a 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 ab15823, 1µg/ml, for 8 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.
  • All lanes : Anti-Histone H4 (acetyl K8) antibody - ChIP Grade (ab15823) at 1 µg/ml

    Lane 1 : Sodium butyrate treated hela histone
    Lane 2 : Untreated hela histone
    Lane 3 : Sodium butyrate treated hela histone with Human Histone H4 (acetyl K8) peptide (ab15824) at 1 µg/ml
    Lane 4 : Untreated hela histone with Human Histone H4 (acetyl K8) peptide (ab15824) at 1 µg/ml
    Lane 5 : Sodium butyrate treated hela histone with Human Histone H4 peptide (ab15825) at 1 µg/ml
    Lane 6 : Untreated hela histone with Human Histone H4 peptide (ab15825) at 1 µg/ml
    Lane 7 : Sodium butyrate treated hela histone with Histone H4 peptide - acetyl K5 (ab15596) at 1 µg/ml
    Lane 8 : Untreated hela histone with Histone H4 peptide - acetyl K5 (ab15596) at 1 µg/ml
    Lane 9 : Sodium butyrate treated hela histone with Human Histone H4 (acetyl K12) peptide (ab15662) at 1 µg/ml
    Lane 10 : Untreated hela histone with Human Histone H4 (acetyl K12) peptide (ab15662) at 1 µg/ml
    Lane 11 : Sodium butyrate treated hela histone with Histone H2A peptide - acetyl K9 at 1 µg/ml
    Lane 12 : Untreated hela histone with Histone H2A peptide - acetyl K9 at 1 µg/ml

    Lysates/proteins at 2.5 µg per lane.

    Secondary
    All lanes : IRDye 680 Conjugated Goat Anti-Rabbit IgG (H+L) at 1/10000 dilution

    Performed under reducing conditions.

    Predicted band size: 11.2 kDa
    Observed band size: 12 kDa
    why is the actual band size different from the predicted?
    Additional bands at: 22 kDa (possible cross reactivity)



    The blot shows that a H4 peptide containing an acetylated K8 residue was able to block recognition of histones by ab15823. Peptides containing other acetylated K residues of H4 and H2A did not block binding of histones by ab15823. This suggests ab15823 specifically recognises Histone H4 acetyl K8.

  • ICC/IF image of ab15823 stained human HeLa cells. The cells were methanol fixed (5 min), permabilised in TBS-T (20 min) and incubated with the antibody (ab15823, 1µg/ml) for 1h at room temperature. 1%BSA / 10% normal goat serum / 0.3M glycine was used to quench autofluorescence and block non-specific protein-protein interactions. 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). DAPI was used to stain the cell nuclei (blue).

  • A: Overall view of a murine rod photoreceptor nucleus immunogold labelled with acetylated H4K8-specific antibodies.
    B: Schematic representation of the H4K8ac-specific labelling. The bar represents 1µm.
  • All lanes : Anti-Histone H4 (acetyl K8) antibody - ChIP Grade (ab15823) at 1 µg/ml

    Lane 1 : Calf Thymus Histone Preparation Nuclear Lysate (ab121)
    Lane 2 : Calf Thymus Histone Preparation Nuclear Lysate (ab121) with Human Histone H4 peptide (ab15825) at 0.5 µg/ml
    Lane 3 : Calf Thymus Histone Preparation Nuclear Lysate (ab121) with Human Histone H4 (acetyl K8) peptide (ab15824) at 0.5 µg/ml
    Lane 4 : Calf Thymus Histone Preparation Nuclear Lysate (ab121) with Histone H4 peptide - acetyl K5 at 0.5 µg/ml
    Lane 5 : Calf Thymus Histone Preparation Nuclear Lysate (ab121) with Human Histone H4 (acetyl K12) peptide (ab15662) at 0.5 µg/ml
    Lane 6 : Calf Thymus Histone Preparation Nuclear Lysate (ab121) with Human Histone H2A (acetyl K9) peptide (ab54016) at 0.5 µg/ml

    Lysates/proteins at 0.5 µg/ml per lane.

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

    Performed under reducing conditions.

    Predicted band size: 11.2 kDa
    Observed band size: 13 kDa why is the actual band size different from the predicted?


    Exposure time: 3 minutes

References

This product has been referenced in:
  • Chu DT  et al. C57BL/6J mice as a polygenic developmental model of diet-induced obesity. Physiol Rep 5:N/A (2017). ChIP ; Mouse . Read more (PubMed: 28400497) »
  • Getun IV  et al. Functional Roles of Acetylated Histone Marks at Mouse Meiotic Recombination Hot Spots. Mol Cell Biol 37:N/A (2017). Read more (PubMed: 27821479) »
See all 39 Publications for this product

Customer reviews and Q&As

1-3 of 3 Abreviews or Q&A

Application
Western blot
Loading amount
20 µg
Gel Running Conditions
Reduced Denaturing (12)
Sample
Human Cell lysate - whole cell (Human HeLa cells)
Specification
Human HeLa cells
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 23°C

Abcam user community

Verified customer

Submitted Nov 13 2013

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunocytochemistry/ Immunofluorescence
Sample
Mouse Cell (retina)
Specification
retina
Fixative
2.5% PAF + 0.1% Glu
Permeabilization
No
Blocking step
BSA as blocking agent for 10 minute(s) · Concentration: 1% · Temperature: 25°C

Caroline Kizilyaprak

Verified customer

Submitted Aug 26 2010

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
ChIP
Sample
Mouse Cell lysate - whole cell (skin epidermis)
Specification
skin epidermis
Type
Cross-linking (X-ChIP)
Duration of cross-linking step: 10 minute(s) and 0 second(s)
Specification of the cross-linking agent: 1% formaldehyde
Detection step
Semiquantitative PCR
Negative control
rabbit IgG immunoprecipitated sample did not show significant PCR amplification.

Abcam user community

Verified customer

Submitted Jan 25 2008

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

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