Recombinant
RabMAb

Anti-Histone H4 (acetyl K8) antibody [EP1002Y] - ChIP Grade (ab45166)

Overview

  • Product name
    Anti-Histone H4 (acetyl K8) antibody [EP1002Y] - ChIP Grade
    See all Histone H4 primary antibodies
  • Description
    Rabbit monoclonal [EP1002Y] to Histone H4 (acetyl K8) - ChIP Grade
  • Host species
    Rabbit
  • Tested applications
    Suitable for: Flow Cyt, WB, IHC-P, ICC/IF, IP, ChIPmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Human
  • Immunogen

    Synthetic peptide within Human Histone H4 aa 1-100 (N terminal) (acetyl K8). The exact sequence is proprietary.
    Database link: P62805

  • Positive control
    • WB- HeLa whole cell lysate +TSA, C6 cell lysate, C6 cell + TSA lysate, NIH/3T3 +TSA whole cell lysate IHC-P: Human normal colon FFPE tissue sections, Mouse kidney paraffin-embedded tissue sections, Rat kidney paraffin-embedded tissue sections. ICC/IF- C6 + TSA lysates
  • General notes

    A trial size is available to purchase for this antibody.

    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

Applications

Our Abpromise guarantee covers the use of ab45166 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.
WB 1/5000 - 1/10000. Predicted molecular weight: 11 kDa.
IHC-P 1/250 - 1/2500. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

See IHC antigen retrieval protocols.

ICC/IF 1/150 - 1/500.
IP 1/20 - 1/50.
ChIP Use 2 µg for 25 µg of chromatin.

Target

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 ab45166 (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). Primers and probes are located in the first kb of the transcribed region.

  • All lanes : Anti-Histone H4 (acetyl K8) antibody [EP1002Y] - ChIP Grade (ab45166) at 1/5000 dilution

    Lane 1 : Untreated HeLa (human cervix adenocarcinoma) whole cell lysate
    Lane 2 : HeLa (human cervix adenocarcinoma) treated with Trichostatin A whole cell lysate

    Lysates/proteins at 10 µg per lane.

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

    Predicted band size: 11 kDa
    Observed band size: 11 kDa



    Blocking and diluting buffer 5% NFDM/TBST
  • Flow Cytometry analysis of HeLa (human cervix adenocarcinoma) treated (Red)/untreated (Green) with 500ng/ml Trichostatin A for 4 hours with purified ab45166 at 1/20 dilution. The secondary antibody was Goat anti rabbit IgG (Alexa Fluor® 488) at 1/2000 dilution. A Rabbit monoclonal IgG (Black) was used as the isotype control and cells without incubation with primary antibody and secondary antibody (Blue) were used as unlabeled control.

  • Immunocytochemistry/immunofluorescence staining of 4% paraformaldehyde fixed; 0.1% triton X 100 permeabilized C6 (rat glioma) cells (non-treated-top panels) and (C6 + TSA(500ng/ml, 4hr)-middle panels) with purified ab45166 at dilution of 1/150. The secondary antibody used was Alexa Fluor® 488; goat anti-rabbit IgG (ab150077) at a dilution of 1/1000. Nucleus was counter-stained with DAPI (blue). ab7291, a mouse anti-tubulin antibody (1/1000) was used to stain tubulin along with ab150120 (AlexaFluor®594 goat anti-mouse secondary, 1/1000) shown in the top right and middle right hand panels. The negative controls are shown in the bottom two panels- for negative control 1 rabbit primary antibody and anti-mouse secondary antibody (ab150120) was used. For negative control 2 mouse primary antibody (ab7291) and anti-rabbit secondary antibody (ab150077) was used.

  • Immunohistochemical staining of paraffin-embedded mouse kidney sections labelling Histone H4 (acetyl K8) with purified ab45166 at dilution of 1:2500. The secondary antibody used was ab97051; a goat anti-rabbit IgG H&L (HRP) at dilution of 1/500. The sample was counter-stained with hematoxylin. Antigen retrieval was performed using EDTA Buffer; pH 9.0. PBS was used instead of the primary antibody as the negative control and is shown in the inset.

  • ab45166 (purified) at 1/20 immunoprecipitating Histone H4 (acetyl K8) in HeLa treated with Trichostatin A whole cell lysate.

    Lane 1 (input): HeLa treated with Trichostatin A whole cell lysate (10µg)

    Lane 2 (+): ab45166 + HeLa treated with Trichostatin A whole cell lysate.

    Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab45166 in HeLa treated with Trichostatin A whole cell lysate.

    For western blotting, ab131366 VeriBlot for IP (HRP) was used as the secondary antibody (1/10000).

    Blocking buffer and concentration: 5% NFDM/TBST.

    Diluting buffer and concentration: 5% NFDM /TBST.



    Lanes 1-2 : Anti-Histone H4 (acetyl K8) antibody [EP1002Y] - ChIP Grade (ab45166) at 1/20 dilution
    Lane 3 : Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) at 1/20 dilution

    Lane 1 : HeLa (human cervix adenocarcinoma) treated with Trichostatin A whole cell lysate at 10 µg
    Lanes 2-3 : HeLa (human cervix adenocarcinoma) treated with Trichostatin A whole cell lysate

    Secondary
    All lanes : VeriBlot for IP Detection Reagent (HRP) (ab131366) at 1/10000 dilution

    Observed band size: 11 kDa
    why is the actual band size different from the predicted?

  • Immunohistochemical staining of paraffin-embedded rat kidney sections labelling Histone H4 (acetyl K8) with purified ab45166 at dilution of 1:2500. The secondary antibody used was ab97051; a goat anti-rabbit IgG H&L (HRP) at dilution of 1/500. The sample was counter-stained with hematoxylin. Antigen retrieval was performed using EDTA Buffer; pH 9.0. PBS was used instead of the primary antibody as the negative control and is shown in the inset.

  • All lanes : Anti-Histone H4 (acetyl K8) antibody [EP1002Y] - ChIP Grade (ab45166) at 1/5000 dilution

    Lane 1 : Untreated C6 (rat glioma) whole cell lysate
    Lane 2 : C6 (rat glioma) treated with Trichostatin A whole cell lysate

    Lysates/proteins at 10 µg per lane.

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

    Predicted band size: 11 kDa
    Observed band size: 11 kDa



    Blocking and diluting buffer 5% NFDM/TBST
  • Immunohistochemical analysis of formalin fixed paraffin embedded human colon tissue sections labelling Histone H4 (acetyl K8) with unpurified ab45166 at dilution of 1/200.

    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 H4 (acetyl K8) antibody [EP1002Y] - ChIP Grade (ab45166) at 1/5000 dilution

    Lane 1 : Untreated NIH/3T3 (mouse embryo) whole cell lysate
    Lane 2 : NIH/3T3 (mouse embryo) treated with Trichostatin A whole cell lysate

    Lysates/proteins at 10 µg per lane.

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

    Predicted band size: 11 kDa
    Observed band size: 11 kDa



    Blocking and diluting buffer 5% NFDM/TBST
  • Immunohistochemical staining of paraffin-embedded human colon sections labelling Histone H4 (acetyl K8) with purified ab45166 at dilution of 1:2500. The secondary antibody used was ab97051; a goat anti-rabbit IgG H&L (HRP) at dilution of 1/500. The sample was counter-stained with hematoxylin. Antigen retrieval was performed using EDTA Buffer; pH 9.0. PBS was used instead of the primary antibody as the negative control and is shown in the inset.

References

This product has been referenced in:
  • Col E  et al. Bromodomain factors of BET family are new essential actors of pericentric heterochromatin transcriptional activation in response to heat shock. Sci Rep 7:5418 (2017). Read more (PubMed: 28710461) »
  • Navarro-Costa P  et al. Early programming of the oocyte epigenome temporally controls late prophase I transcription and chromatin remodelling. Nat Commun 7:12331 (2016). IF ; Drosophila melanogaster . Read more (PubMed: 27507044) »
See all 6 Publications for this product

Customer reviews and Q&As

Application
ChIP
Sample
Human Cell lysate - whole cell (THP-1 cells)
Negative control
gene desert region, no antibody control
Specification
THP-1 cells
Detection step
Real-time PCR
Type
Cross-linking (X-ChIP)
Duration of cross-linking step: 10 minute(s) and 0 second(s)
Specification of the cross-linking agent: formaldehyde
Positive control
active gene promoter

Abcam user community

Verified customer

Submitted Oct 17 2018

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