Anti-Histone H4 (unmodified K20) antibody [EPR22116] - ChIP Grade

9 product images

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  Immunoprecipitation - Anti-Histone H4 (unmodified K20) antibody [EPR22116] - ChIP Grade (ab227804)

  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) (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 (Rabbit IgG, monoclonal [EPR25A] - Isotype Control 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: Immunoprecipitation - Anti-Histone H4 (unmodified K20) antibody [EPR22116] - ChIP Grade (ab227804)

  Predicted band size: 11 kDa

  Observed band size: 12 kDa

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  Western blot - Anti-Histone H4 (unmodified K20) antibody [EPR22116] - ChIP Grade (ab227804)

  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.

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

  Lane 1: SET8 depleted lysate from U-2 OS (human bone osteosarcoma epithelial cell line), whole cell lysate at 20 µg

  Lane 2: G1-arrested lysate from U-2 OS whole cell lysate at 20 µg

  Lane 3: HeLa (human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 20 µg

  Lane 4: NIH/3T3 (mouse embryo fibroblast cell line) whole cell lysate at 20 µg

  Secondary

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

  Predicted band size: 11 kDa

  Observed band size: 12 kDa

  Exposure time: 3min

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H4 (unmodified K20) antibody [EPR22116] - ChIP Grade (ab227804)

  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 Antigen Retrieval Buffer (100X Tris-EDTA Buffer, pH 9.0) ab93684 (Tris/EDTA buffer, pH 9.0).

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  Western blot - Anti-Histone H4 (unmodified K20) antibody [EPR22116] - ChIP Grade (ab227804)

  Blocking and dilution buffer: 5% NFDM/TBST.
  

  The recombinant proteins were His-tagged.

  All lanes: Western blot - Anti-Histone H4 (unmodified K20) antibody [EPR22116] - ChIP Grade (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: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/50000 dilution

  Predicted band size: 11 kDa

  Observed band size: 12 kDa

  Exposure time: 3min

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

  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).

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H4 (unmodified K20) antibody [EPR22116] - ChIP Grade (ab227804)

  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 Antigen Retrieval Buffer (100X Tris-EDTA Buffer, pH 9.0) ab93684 (Tris/EDTA buffer, pH 9.0).

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H4 (unmodified K20) antibody [EPR22116] - ChIP Grade (ab227804)

  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 Antigen Retrieval Buffer (100X Tris-EDTA Buffer, pH 9.0) ab93684 (Tris/EDTA buffer, pH 9.0).

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H4 (unmodified K20) antibody [EPR22116] - ChIP Grade (ab227804)

  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 Antigen Retrieval Buffer (100X Tris-EDTA Buffer, pH 9.0) ab93684 (Tris/EDTA buffer, pH 9.0).

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  Peptide Array - Anti-Histone H4 (unmodified K20) antibody [EPR22116] - ChIP Grade (ab227804)

  ab227804 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, is available under the Support & downloads section.