Recombinant
RabMAb

Anti-Histone H4 antibody [EPR16599] - ChIP Grade (ab177840)

Overview

  • Product name
    Anti-Histone H4 antibody [EPR16599] - ChIP Grade
    See all Histone H4 primary antibodies
  • Description
    Rabbit monoclonal [EPR16599] to Histone H4 - ChIP Grade
  • Tested applications
    Suitable for: PepArr, ChIP, IHC-P, WB, ICC/IFmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Human, Drosophila melanogaster
    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 H4 aa 50 to the C-terminus. The exact sequence is proprietary.
    Database link: P62805

  • Positive control
    • WB: HeLa and NIH/3T3 whole cell lysates; Drosophila whole lysate. ICC/IF: HeLa cells. IHC-P: Human colon, mouse pancreas and rat cerebral cortex tissues. ChIP: Chromatin from HeLa cells.
  • General notes

    This product is a recombinant rabbit monoclonal antibody.

    Alternative versions available:

    Anti-Histone H4 antibody (Alexa Fluor® 647) [EPR16599] (ab207075)

    Anti-Histone H4 antibody (Alexa Fluor® 488) [EPR16599] (ab207387)

    Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMab® patents

Properties

Applications

Our Abpromise guarantee covers the use of ab177840 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
PepArr Use at an assay dependent concentration.
ChIP Use 2 µg for 25 µg of chromatin.
IHC-P 1/2000. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
WB 1/1000. Detects a band of approximately 11 kDa (predicted molecular weight: 11 kDa).

We recommend using 3% milk as the blocking agent for Western blot.

ICC/IF 1/100.

Target

Images

  • All lanes : Anti-Histone H4 antibody [EPR16599] - ChIP Grade (ab177840) at 1/1000 dilution

    Lane 1 : Histone H1 Recombinant Protein
    Lane 2 : Histone H2A Recombinant Protein
    Lane 3 : Histone H2B Recombinant Protein
    Lane 4 : Histone H3.1 Recombinant Protein
    Lane 5 : Histone H4 Recombinant Protein
    Lane 6 : Histone H1 Recombinant Protein
    Lane 7 : Histone H2A Recombinant Protein
    Lane 8 : Histone H2B Recombinant Protein
    Lane 9 : Histone H3.1 Recombinant Protein
    Lane 10 : Histone H4 Recombinant Protein

    Lysates/proteins at 0.1 µg per lane.

    Secondary
    Goat Anti-Rabbit IgG (H+L), Peroxidase Conjugated at 1/50000 dilution
    Developed using the ECL technique

    Performed under reducing conditions.

    Predicted band size : 11 kDa
    Observed band size : 15 kDa (why is the actual band size different from the predicted?)


    Exposure time : 4 minutes

    Lanes 1-5: 1% BSA blocking buffer

    Lanes 6-10: 3% Milk blocking buffer

     

    We recommend using 3% milk as the blocking agent for Western blot.

  • Chromatin was prepared from Hela cells according to the Abcam X-ChIP protocol. Cells were fixed with 0.75% formaldehyde for 10min. The ChIP was performed with 25μg of chromatin, 2μg of ab177840 (blue), and 20μl of Anti rabbit IgG sepharose beads. 2μg of rabbit normal IgG was added to the beads control (yellow). The immunoprecipitated DNA was quantified by real time PCR (SYBR approach). Primers are located in the first kb of the transcribed region.

  • Anti-Histone H4 antibody [EPR16599] - ChIP Grade (ab177840) at 1/1000 dilution + Drosophila whole lysates at 10 µg

    Secondary
    Goat Anti-Rabbit IgG, (H+L),Peroxidase conjugated at 1/1000 dilution
    Developed using the ECL technique

    Performed under reducing conditions.

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


    Exposure time : 3 minutes

    Blocking/Dilution buffer: 5% NFDM/TBST.

  • All batches of ab177840 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.

  • All lanes : Anti-Histone H4 antibody [EPR16599] - ChIP Grade (ab177840) at 1/5000 dilution

    Lane 1 : HeLa whole cell lysates
    Lane 2 : NIH/3T3 whole cell lysates

    Lysates/proteins at 10 µg per lane.

    Secondary
    Goat Anti-Rabbit IgG, (H+L),Peroxidase conjugated at 1/1000 dilution
    Developed using the ECL technique

    Performed under reducing conditions.

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


    Exposure time : 3 minutes

    Blocking/Dilution buffer: 5% NFDM/TBST.

  • Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa cells labeling Histone H4 with ab177840 at 1/100 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/400 dilution (green). Nuclear staining on HeLa cell line is observed. The nuclear counter stain is DAPI (blue). Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/500 dilution and ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution (red).
    The negative controls are as follows:
    1. ab177840 at 1/100 dilution followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution.
    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.

  • Immunohistochemical analysis of paraffin-embedded Human colon tissue labeling Histone H4 with ab177840 at 1/2000 dilution, followed by prediluted Goat Anti-Rabbit IgG H&L (HRP). Nucleus staining on glandular epithelium of Human colon tissue is observed. Counter stained with Hematoxylin. 

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

  • Immunohistochemical analysis of paraffin-embedded Mouse pancreas tissue labeling Histone H4 with ab177840 at 1/2000 dilution, followed by prediluted Goat Anti-Rabbit IgG H&L (HRP). Nucleus staining on glandular epithelium of mouse pancreas tissue is observed. Counter stained with Hematoxylin.

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

  • Immunohistochemical analysis of paraffin-embedded Rat cerebral cortex tissue labeling Histone H4 with ab177840 at 1/2000 dilution, followed by prediluted Goat Anti-Rabbit IgG H&L (HRP). Nuclear staining on neuron cells of cerebral cortex tissue is observed. Counter stained with Hematoxylin.

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

References

This product has been referenced in:

See 1 Publication for this product

Customer reviews and Q&As

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Human Cell lysate - nuclear (MCF7 cells)
Gel Running Conditions
Reduced Denaturing (15%)
Loading amount
10 µg
Treatment
20ng/ml TNF alpha
Specification
MCF7 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

Application
Western blot
Sample
Human Recombinant protein (Recombinant octamers & purified K-562 cell histone)
Gel Running Conditions
Reduced Denaturing (18% gel)
Loading amount
0.5 µg
Specification
Recombinant octamers & purified K-562 cell histone
Blocking step
Odyssey blocking solution 1/1 (vol/vol) PBS as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 50% · Temperature: 25°C
Username

Dr. Ragnhild Eskeland

Verified customer

Submitted May 11 2015

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

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