Recombinant
RabMAb

Recombinant Anti-Histone H4 (unmodified K20) antibody [EPR22116] (ab227804)

Overview

  • Product name

    Anti-Histone H4 (unmodified K20) antibody [EPR22116]
    See all Histone H4 primary antibodies
  • Description

    Rabbit monoclonal [EPR22116] to Histone H4 (unmodified K20)
  • Host species

    Rabbit
  • Specificity

    Peptide array data showed that this RabMAb bound three methylated peptides (R19me1, R19me2a and R23me1) but with lower affinity than the unmethylated peptide.
  • Tested applications

    Suitable for: ChIP, IP, IHC-P, WBmore details
  • Species reactivity

    Reacts with: Mouse, Rat, Human
    Predicted to work with: Cow, Caenorhabditis elegans, Drosophila melanogaster, Schizosaccharomyces pombe
  • Immunogen

    Synthetic peptide within Human Histone H4 aa 1-100. The exact sequence is proprietary.
    Database link: P62805

  • Positive control

    • WB: SET8 depleted lysate from U-2 OS cells; HeLa and NIH/3T3 whole cell lysates; Unmodified recombinant H4 protein. IP: SET8 depleted lysate from U-2 OS cells. IHC-P: Human colon and bladder cancer tissue; Mouse spleen tissue; Rat spleen tissue. ChIP: Chromatin prepared 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: 0.05% BSA, 40% Glycerol, PBS
  • Concentration information loading...
  • Purity

    Protein A purified
  • Clonality

    Monoclonal
  • Clone number

    EPR22116
  • Isotype

    IgG

Applications

Our Abpromise guarantee covers the use of ab227804 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
ChIP Use 2 µg for 25 µg of chromatin.
IP 1/30.
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 12 kDa (predicted molecular weight: 11 kDa).

Target

Images

  • All lanes : Anti-Histone H4 (unmodified K20) antibody [EPR22116] (ab227804) at 1/1000 dilution

    Lane 1 : SET8 depleted lysate from U-2 OS (human bone osteosarcoma epithelial cell line), whole cell lysate
    Lane 2 : G1-arrested lysate from U-2 OS whole cell lysate
    Lane 3 : HeLa (human epithelial cell line from cervix adenocarcinoma) whole cell lysate
    Lane 4 : NIH/3T3 (mouse embryo fibroblast cell line) whole cell lysate

    Lysates/proteins at 20 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

    Predicted band size: 11 kDa
    Observed band size: 12 kDa
    why is the actual band size different from the predicted?


    Exposure time: 3 minutes


    Blocking and dilution buffer: 5% NFDM/TBST.

    Depletion of Set8, a histone methyltransferase, delays S-phase progression and impairs DNA replication (PMID: 18166648). It also prevents the monomethylation of H4K20, which is normally mediated by Set8 (PMID: 18166648).

    The lysates in lanes 1 & 2 were kindly provided by our collaborator Dr. Anja Groth, Biotech Research and Innovation Centre (BRIC). University of Copenhagen.

  • Immunohistochemical analysis of paraffin-embedded human colon tissue labeling Histone H4K20 with ab227804 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) ready to use. Sporadic nuclear staining in human colon tissue is observed. Counter stained with Hematoxylin.

    Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) ready to use.

    Heat-mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

  • Histone H4 (unmodified K20) was immunoprecipitated from 0.35 mg of SET8 depleted U-2 OS (human bone osteosarcoma epithelial cell line) whole cell lysate with ab227804 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab227804 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/5000 dilution.

    Lane 1: SET8 depleted U-2 OS whole cell lysate 10 μg (Input).

    Lane 2: ab227804 IP in SET8 depleted U-2 OS whole cell lysate.

    Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab227804 in SET8 depleted U-2 OS whole cell lysate.

    Blocking and dilution buffer and concentration: 5% NFDM/TBST.
    Exposure time: 30 seconds.

    Depletion of Set8, a histone methyltransferase, delays S-phase progression and impairs DNA replication (PMID: 18166648). It also prevents the monomethylation of H4K20, which is normally mediated by Set8 (PMID: 18166648).

    This lysate was kindly provided by our collaborator Dr. Anja Groth, Biotech Research and Innovation Centre (BRIC). University of Copenhagen.

  • All lanes : Anti-Histone H4 (unmodified K20) antibody [EPR22116] (ab227804) at 1/1000 dilution

    Lane 1 : Unmodified recombinant H4 protein, 10 ng
    Lane 2 : Recombinant mutant H4 chemically modified to mimic H4K20Me1, 10 ng
    Lane 3 : Recombinant mutant H4 chemically modified to mimic H4K20Me2, 10 ng
    Lane 4 : Recombinant mutant H4 chemically modified to mimic H4K20Me3, 10 ng
    Lane 5 : Unmodified recombinant mutant H4 (K20 mutated to C), 10 ng

    Secondary
    All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/50000 dilution

    Predicted band size: 11 kDa
    Observed band size: 12 kDa why is the actual band size different from the predicted?


    Exposure time: 3 minutes


    Blocking and dilution buffer: 5% NFDM/TBST.

    The recombinant proteins were His-tagged.

     

  • Immunohistochemical analysis of paraffin-embedded rat spleen tissue labeling Histone H4K20 with ab227804 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) ready to use. Sporadic nuclear staining in rat spleen is observed. Counter stained with Hematoxylin.

    Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) ready to use.

    Heat-mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

  • Immunohistochemical analysis of paraffin-embedded mouse spleen tissue labeling Histone H4K20 with ab227804 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) ready to use. Nuclear staining in mouse spleen is observed. Counter stained with Hematoxylin.

    Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) ready to use.

    Heat-mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

  • Immunohistochemical analysis of paraffin-embedded human bladder cancer tissue labeling Histone H4K20 with ab227804 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) ready to use. Sporadic nuclear staining in human bladder cancer is observed. Counter stained with Hematoxylin.

    Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) ready to use.

    Heat-mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

  • Chromatin was prepared from HeLa cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10min. The ChIP was performed with 25 μg of chromatin, 2 μg of ab227804 (red), and 20 µl of protein A/G sepharose beads slurry (10 µl of sepharose A beads + 10 µl of sepharose G beads). 2 μg of rabbit normal IgG was added to the beads control (gray). The immunoprecipitated DNA was quantified by real time PCR (SYBR approach).

References

This product has been referenced in:

  • Nakamura K  et al. H4K20me0 recognition by BRCA1-BARD1 directs homologous recombination to sister chromatids. Nat Cell Biol N/A:N/A (2019). IF . Read more (PubMed: 30804502) »
See 1 Publication for this product

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