Recombinant
RabMAb

Recombinant 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
  • Host species

    Rabbit
  • 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

    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

  • Form

    Liquid
  • Storage instructions

    Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C long term. Avoid freeze / thaw cycle.
  • Storage buffer

    Preservative: 0.01% Sodium azide
    Constituents: 59% PBS, 40% Glycerol, 0.05% BSA
  • Concentration information loading...
  • Purity

    Protein A purified
  • Clonality

    Monoclonal
  • Clone number

    EPR16599
  • Isotype

    IgG
  • Research areas

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

  • 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.

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

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

    Lysates/proteins at 0.1 µg per lane.

    Secondary
    All lanes : 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.

  • 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 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
    All lanes : 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.

  • ab177840 was 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.

  • 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.

    Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

  • 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.

    Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

  • 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.

    Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

References

This product has been referenced in:

  • Lu Y  et al. Cohesin acetyltransferase Esco2 regulates SAC and kinetochore functions via maintaining H4K16 acetylation during mouse oocyte meiosis. Nucleic Acids Res 45:9388-9397 (2017). Read more (PubMed: 28934466) »
  • Bagheri-Sereshki N  et al. The Effects of Chemotherapeutic Agents, Bleomycin, Etoposide, and Cisplatin, on Chromatin Remodeling in Male Rat Germ Cells. Biol Reprod 94:81 (2016). Read more (PubMed: 26911428) »
See all 3 Publications for this product

Customer reviews and Q&As

1-3 of 3 Abreviews or Q&A

Application
Western blot
Sample
Mouse Cell lysate - whole cell (mouse embryonic stem cells (J1))
Gel Running Conditions
Reduced Denaturing
Loading amount
20 µg
Specification
mouse embryonic stem cells (J1)
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 22°C

Abcam user community

Verified customer

Submitted Nov 02 2018

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

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

Dr. Ragnhild Eskeland

Verified customer

Submitted May 11 2015

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