Anti-Histone H3 (di methyl K4) antibody [Y47] - ChIP Grade

16 product images

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  ChIP - Anti-Histone H3 (di methyl K4) antibody [Y47] - ChIP Grade (ab32356)

  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, 2μg of ab32356 (red), and 20μl of Anti Rabbit IgG sepharose beads. 2μg of rabbit normal IgG was added to the beads control (grey). The immunoprecipitated DNA was quantified by real time PCR (Sybr green approach). Primers and probes are located in the first kb of the transcribed region.

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  Immunoprecipitation - Anti-Histone H3 (di methyl K4) antibody [Y47] - ChIP Grade (ab32356)

  ab32356 (purified) at 1/30 dilution (20 μg/ml) immunoprecipitating Histone H3 di methyl K4 in HepG2 whole cell lysate.
  
  Lane 1 (input): HepG2 (human hepatocellular carcinoma epithelial cell) whole cell lysate 10μg
  Lane 2 (+): ab32356 & HepG2 whole cell lysate
  Lane 3 (-): Rabbit monoclonal IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) instead of ab32356 in HepG2 whole cell lysate
  For western blotting, ab32356 at 1/500 and veriBlot for IP secondary antibody (HRP) (VeriBlot for IP Detection Reagent (HRP) ab131366) was used at 1/1000 dilution.
  
  Blocking and diluting buffer: 5% NFDM /TBST.

  All lanes: Immunoprecipitation - Anti-Histone H3 (di methyl K4) antibody [Y47] - ChIP Grade (ab32356)

  Predicted band size: 15 kDa

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  ChIP-sequencing - Anti-Histone H3 (di methyl K4) antibody [Y47] - ChIP Grade (ab32356)

  Chromatin was prepared from HeLa cells. Cells were fixed with 1% formaldehyde for 10 minutes. ChIP was performed with 107 cells and 4 μg of Anti-Histone H3 (di methyl K4) antibody [Y47] - ChIP Grade (ab32356). ChIP DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 30 million reads.
  Additional screenshots of mapped reads can be downloaded here.

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  Immunocytochemistry/ Immunofluorescence - Anti-Histone H3 (di methyl K4) antibody [Y47] - ChIP Grade (ab32356)

  Immunocytochemistry/Immunofluorescence analysis of HepG2 cells labelling Histone H3 (di methyl K4) with purified ab32356 at a dilution of 1/1000. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody. The cells were co-stained with Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291, a mouse anti-tubulin (1/1000) using Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/1000) as the secondary antibody. Nuclei counterstained with DAPI (blue).
  

  Control 1: primary antibody (1/1000) and secondary antibody, Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/1000).

  Control 2: Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291 (1/1000) and secondary antibody, Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000).

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  Flow Cytometry (Intracellular) - Anti-Histone H3 (di methyl K4) antibody [Y47] - ChIP Grade (ab32356)

  Intracellular Flow Cytometry analysis of HepG2 (human hepatocellular carcinomal) cells labeling Histone H3 di methyl K4 with purified ab32356 at 1/500 dilution (1.17μg/ml) (Red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. A Goat anti rabbit IgG (Alexa Fluor^® 488, Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077) secondary antibody was used at 1/2000 dilution. Isotype control - Rabbit monoclonal IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) / Black. Unlabeled control - Unlabelled cells / blue.
  

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  Western blot - Anti-Histone H3 (di methyl K4) antibody [Y47] - ChIP Grade (ab32356)

  All lanes: Western blot - Anti-Histone H3 (di methyl K4) antibody [Y47] - ChIP Grade (ab32356) at 1/10000 dilution

  Lane 1: HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 20 µg

  Lane 2: HEK-293 (Human epithelial cell line from embryonic kidney) whole cell lysate at 20 µg

  Lane 3: SH-SY5Y (Human neuroblastoma cell line from bone marrow) whole cell lysate at 20 µg

  Lane 4: C6 (Rat glial tumor cell line) whole cell lysate at 20 µg

  Secondary

  All lanes: Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

  Predicted band size: 15 kDa

  Observed band size: 17 kDa

  Exposure time: 5s

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H3 (di methyl K4) antibody [Y47] - ChIP Grade (ab32356)

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of mouse colon tissue labelling Histone H3 (di methyl K4) with purified ab32356 at a dilution of 1/800. Antigen retrieval was performed using Tris/EDTA buffer, pH9. Goat Anti-Rabbit IgG H&L (HRP) ab97051, a HRP-conjugated goat anti-rabbit IgG H&L was used as the secondary antibody (1/500). Counter stained with hematoxylin.

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  Western blot - Anti-Histone H3 (di methyl K4) antibody [Y47] - ChIP Grade (ab32356)

  All lanes: Western blot - Anti-Histone H3 (di methyl K4) antibody [Y47] - ChIP Grade (ab32356) at 1/2000 dilution

  Lane 1: L6 (Rat skeletal muscle cell line) whole cell lysate at 20 µg

  Lane 2: NIH/3T3 (Mouse embryonic fibroblast cell line) whole cell lysate at 20 µg

  Lane 3: COS-1 (African green monkey kidney fibroblast-like cell line) whole cell lysate at 20 µg

  Lane 4: UMNSAH/DF-1 (Transformed chicken embryonic fibroblast cell line) whole cell lysate at 20 µg

  Lane 5: MDBK (Bovine kidney 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 1/1000 dilution

  Predicted band size: 15 kDa

  Observed band size: 17 kDa

  Exposure time: 5s

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  Flow Cytometry (Intracellular) - Anti-Histone H3 (di methyl K4) antibody [Y47] - ChIP Grade (ab32356)

  Intracellular Flow Cytometry analysis of HeLa cells labelling Histone H3 (di methyl K4) with purified ab32356 at 1/150 (red). Cells were fixed with 80% methanol. An Alexa Fluorr^® 488-conjugated goat anti-rabbit IgG (1/500) was used as the secondary antibody. Black - Isotype control, rabbit monoclonal IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730). Blue - Unlabelled control, cells without incubation with primary and secondary antibodies.
  

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H3 (di methyl K4) antibody [Y47] - ChIP Grade (ab32356)

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of rat spleen tissue labelling Histone H3 (di methyl K4) with purified ab32356 at a dilution of 1/800. Antigen retrieval was performed using Tris/EDTA buffer, pH9. Goat Anti-Rabbit IgG H&L (HRP) ab97051, a HRP-conjugated goat anti-rabbit IgG H&L was used as the secondary antibody (1/500). Counter stained with hematoxylin.

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  Flow Cytometry (Intracellular) - Anti-Histone H3 (di methyl K4) antibody [Y47] - ChIP Grade (ab32356)

  Overlay histogram showing HeLa cells stained with unpurified ab32356 (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 (ab32356, 1/100 dilution) for 30 min at 22°C. The secondary antibody used was a goat anti-rabbit DyLight^® 488 (IgG; H+L) (Goat Anti-Rabbit IgG H&L (DyLight® 488) preadsorbed ab96899) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1μg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed.
  

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H3 (di methyl K4) antibody [Y47] - ChIP Grade (ab32356)

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human cervical carcinoma tissue labelling Histone H3 (di methyl K4) with purified ab32356 at a dilution of 1/800. Antigen retrieval was performed using Tris/EDTA buffer, pH9. Goat Anti-Rabbit IgG H&L (HRP) ab97051, a HRP-conjugated goat anti-rabbit IgG H&L was used as the secondary antibody (1/500). Counter stained with hematoxylin.

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  Immunocytochemistry/ Immunofluorescence - Anti-Histone H3 (di methyl K4) antibody [Y47] - ChIP Grade (ab32356)

  ICC/IF image of unpurified ab32356 stained HepG2 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 (ab32356 , 5μg/ml) overnight at +4°C. The secondary antibody (green) was a goat anti-rabbit DyLight® 488 (IgG - H&L, pre-adsorbed) (Goat Anti-Rabbit IgG H&L (DyLight® 488) preadsorbed ab96899) used at a 1/250 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.
  
  

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  ChIP-sequencing - Anti-Histone H3 (di methyl K4) antibody [Y47] - ChIP Grade (ab32356)

  Chromatin was prepared from HeLa cells. Cells were fixed with 1% formaldehyde for 10 minutes. ChIP was performed with 30 µg of chromatin and 4 µg of Anti-Histone H3 (di methyl K4) antibody [Y47] - ChIP Grade (ab32356). ChIP DNA was sequenced on the Illumina NextSeq 500 to a depth of 30 million reads. ChIP-Seq validation performed by Active Motif, Carlsbad, CA.
  Additional screenshots of mapped reads can be downloaded here.

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  ChIC/CUT&RUN sequencing - Anti-Histone H3 (di methyl K4) antibody [Y47] - ChIP Grade (ab32356)

  ChIC/CUT&RUN was performed using a pAG-MNAse at a final concentration of 700 ng/mL, 2.5 x 10^5 HeLa (Human cervix adenocarcinoma epithelial cell line) cells and 5 µg of ab32356 [Y47]. The resulting DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 10 million reads. The negative IgG control Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730 is also shown.
  Additional screenshots of mapped reads can be found in the Protocol booklet in the Support and downloads section.
  The University of Geneva owns patents relevant to ChIC (Chromatin Immuno-Cleavage) methods.

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  Peptide Array - Anti-Histone H3 (di methyl K4) antibody [Y47] - ChIP Grade (ab32356)

  ab32356 or Anti-Histone H3 (di methyl K4) antibody [EPR17707] - ChIP Grade ab176878 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 the area under the curve when antibody binding values are plotted against the corresponding peptide concentration. Each circle area is normalized to the peptide with the strongest affinity. Peptide Array, ab32356 binds to Histone H3 (di methyl K4) on Peptide Array but shows some weak non-specific binding to other Histone H3 modifications. We recommend Anti-Histone H3 (di methyl K4) antibody [EPR17707] - ChIP Grade ab176878 for Peptide Array of Histone H3 (di methyl K4).