Anti-Histone H4 antibody [mAbcam 31830] - ChIP Grade – BSA and Azide free (ab238663)

Overview

  • Product name

    Anti-Histone H4 antibody [mAbcam 31830] - ChIP Grade – BSA and Azide free
    See all Histone H4 primary antibodies
  • Description

    Mouse monoclonal [mAbcam 31830] to Histone H4 - ChIP Grade – BSA and Azide free
  • Host species

    Mouse
  • Tested applications

    Suitable for: WB, IP, IHC-P, Flow Cyt, ChIP, ICC/IFmore details
  • Species reactivity

    Reacts with: Cow, Human
    Predicted to work with: Mouse, Rat
  • Immunogen

    Synthetic peptide corresponding to Human Histone H4 aa 50 to the C-terminus (C terminal) conjugated to keyhole limpet haemocyanin.
    Database link: P62805
    (Peptide available as ab13843)

  • Positive control

    • This antibody gave a positive signal in the following lysates: Calf Thymus Histone Preparation Nuclear Lysate HeLa Histone Preparation Nuclear Lysate Histone H4 Recombinant Protein
  • General notes

    Ab238663 is a PBS-only buffer format of ab31830. Please refer to ab31830  for recommended dilutions, protocols, and image data.

    This antibody clone is manufactured by Abcam.

    If you require this antibody in a particular buffer formulation or a particular conjugate for your experiments, please contact orders@abcam.com or you can find further information here.

Properties

  • Form

    Liquid
  • Storage instructions

    Shipped at 4°C. Store at +4°C. Do Not Freeze.
  • Storage buffer

    pH: 7.50
    Constituent: PBS
  • Concentration information loading...
  • Purity

    IgG fraction
  • Clonality

    Monoclonal
  • Clone number

    mAbcam 31830
  • Myeloma

    Sp2/0-Ag14
  • Isotype

    IgG1
  • Light chain type

    kappa

Applications

Our Abpromise guarantee covers the use of ab238663 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
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).
IP Use at an assay dependent concentration.
IHC-P Use a concentration of 0.05 - 1 µg/ml. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
Flow Cyt Use 1µg for 106 cells.

ab170190 - Mouse monoclonal IgG1, is suitable for use as an isotype control with this antibody.

 

ChIP Use 5 µg for 25 µg of chromatin.
ICC/IF Use a concentration of 5 µg/ml.

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, 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.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, L-Arginine and sodium azide (ab31830).

  • 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.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, L-Arginine and sodium azide (ab31830).

     

  • 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.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, L-Arginine and sodium azide (ab31830).

  • IHC image of Histone H4 staining in human breast fibroadenoma 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, 0.1µg/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.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, L-Arginine and sodium azide (ab31830).

  • 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.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, L-Arginine and sodium azide (ab31830).

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

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

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

    Performed under reducing conditions.

    Predicted band size: 14 kDa



    This data was developed using the same antibody clone in a different buffer formulation containing PBS, L-Arginine and sodium azide (ab31830).

  • All lanes : Anti-Histone H4 antibody [mAbcam 31830] - ChIP Grade (ab31830) at 5 µg/ml

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

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

    Performed under reducing conditions.

    Predicted band size: 14 kDa


    Exposure time: 2 minutes


    This data was developed using the same antibody clone in a different buffer formulation containing PBS, L-Arginine and sodium azide (ab31830).

References

ab238663 has not yet been referenced specifically in any publications.

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