Anti-Histone H4 antibody [mAbcam 31830] - ChIP Grade (ab31830)

Overview

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 bufferPreservative: 0.02% Sodium Azide
    Constituents: PBS, pH 7.5
  • Concentration information loading...
  • PurityIgG fraction
  • ClonalityMonoclonal
  • Clone numbermAbcam 31830
  • MyelomaSp2/0-Ag14
  • IsotypeIgG1
  • Light chain typekappa
  • Research areas

Applications

Our Abpromise guarantee covers the use of ab31830 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 1µg for 106 cells. ab170190-Mouse monoclonal IgG1, is suitable for use as an isotype control with this antibody.
IHC-P Use a concentration of 0.05 - 1 µg/ml. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
IP Use at an assay dependent concentration.
WB Use a concentration of 1 µg/ml. Detects a band of approximately 13 kDa (predicted molecular weight: 14 kDa).Can be blocked with Human Histone H4 peptide (ab13843).
ChIP Use 5 µg for 25 µg of chromatin.
ICC/IF Use a concentration of 5 µg/ml.

Target

Anti-Histone H4 antibody [mAbcam 31830] - ChIP Grade 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, 5µg of ab31830 (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 for active and inactive loci, Sybr green approach for heterochromatic loci). Primers and probes are located in the first kb of the transcribed region.

  • Overlay histogram showing HeLa cells stained with ab31830 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab31830, 1µg/1x106 cells) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed.

  • All lanes : Anti-Histone H4 antibody [mAbcam 31830] - ChIP Grade (ab31830) at 1 µg/ml

    Lane 1 : Calf Thymus Histone Preparation Nuclear Lysate at 0.5 µg
    Lane 2 : HeLa Histone Preparation Nuclear Lysate at 2.5 µg
    Lane 3 : Histone H4 Recombinant Protein at 0.1 µg
    Lane 4 : Histone H3.1 Recombinant Protein at 0.1 µg

    Secondary
    Goat polyclonal to Mouse IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

    Performed under reducing conditions.

    Predicted band size : 14 kDa
    Observed band size : 13 kDa (why is the actual band size different from the predicted?)
  • All lanes : Anti-Histone H4 antibody [mAbcam 31830] - ChIP Grade (ab31830) at 5 µg/ml

    Lane 1 : Calf Thymus Histone Preparation Nuclear Lysate at 0.5 µg
    Lane 2 : HeLa Histone Preparation Nuclear Lysate at 2.5 µg
    Lane 3 : Histone H4 Recombinant Protein at 0.1 µg
    Lane 4 : Histone H3.1 Recombinant Protein at 0.1 µg
    Lane 5 : Histone H2A Recombinant Protein at 0.1 µg
    Lane 6 : Histone H2B Recombinant Protein at 0.1 µg
    Lane 7 : Histone H1 Recombinant Protein at 0.1 µg
    Lane 8 : Calf Thymus Histone Preparation Nuclear Lysate at 0.5 µg with Human Histone H4 peptide (ab13843) at 5 µg/ml
    Lane 9 : HeLa Histone Preparation Nuclear Lysate at 2.5 µg with Human Histone H4 peptide (ab13843) at 5 µg/ml
    Lane 10 : Histone H4 Recombinant Protein at 0.1 µg with Human Histone H4 peptide (ab13843) at 5 µg/ml
    Lane 11 : Histone H3.1 Recombinant Protein at 0.1 µg with Human Histone H4 peptide (ab13843) at 5 µg/ml
    Lane 12 : Histone H2A Recombinant Protein at 0.1 µg with Human Histone H4 peptide (ab13843) at 5 µg/ml
    Lane 13 : Histone H2B Recombinant Protein at 0.1 µg with Human Histone H4 peptide (ab13843) at 5 µg/ml
    Lane 14 : Histone H1 Recombinant Protein at 0.1 µg with Human Histone H4 peptide (ab13843) at 5 µg/ml

    Secondary
    Goat polyclonal to Mouse IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

    Performed under reducing conditions.

    Predicted band size : 14 kDa
    Observed band size : 13 kDa (why is the actual band size different from the predicted?)


    Exposure time : 2 minutes
  • Histone H4 was immunoprecipitated using 0.5mg Hela whole cell extract, 10µg of Mouse monoclonal to Histone H4 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 70oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab31830.
    Secondary: Protein G-HRP at 1/500 dilution.
    Band: 13kDa: Histone H4; non specific - 25 and 55kDa: We are unsure as to the identity of this extra band.
  • IHC image of Histone H4 staining in human cervix carcinoma formalin fixed paraffin embedded tissue section, performed on a Leica 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 ab31380, 1µg/ml, for 15 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.
  • ICC/IF image of ab31830 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 (ab31830, 5µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-mouse 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.

References for Anti-Histone H4 antibody [mAbcam 31830] - ChIP Grade (ab31830)

This product has been referenced in:
  • Wu JL  et al. Cloning and Functional Analysis of Histones H3 and H4 in Nuclear Shaping during Spermatogenesis of the Chinese Mitten Crab, Eriocheir sinensis. PLoS One 10:e0126623 (2015). Read more (PubMed: 25993499) »
  • Westman J  et al. Extracellular Histones Induce Chemokine Production in Whole Blood Ex Vivo and Leukocyte Recruitment In Vivo. PLoS Pathog 11:e1005319 (2015). Mouse . Read more (PubMed: 26646682) »

See all 8 Publications for this product

Product Wall

Application Western blot
Sample Xenopus laevis Tissue lysate - other (stage 18 lane 1,2: cytosol lane 3,4: chromatin fra)
Gel Running Conditions Reduced Denaturing (15%)
Loading amount 0 cells
Specification stage 18 lane 1,2: cytosol lane 3,4: chromatin fra
Blocking step BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 2% · Temperature: 21°C
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Submitted Aug 07 2015



Bezüglich des Immunogens kann ich Ihnen sagen, dass genau die selbe Sequenz verwendet wurde. Daher sind diese Antikörper in Kombination also unbrauchbar für einen "sandwich immunoassay".

Um Ihnen helfen zu k&...

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