Anti-Histone H3 (mono methyl K4) antibody [ERP16597] - ChIP Grade

10 product images

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  ChIP - Anti-Histone H3 (mono methyl K4) antibody [ERP16597] - ChIP Grade (ab176877)

  Chromatin was prepared from HeLa (human epithelial cell line from cervix adenocarcinoma) cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10 min.
  

  The ChIP was performed with 25 μg of chromatin, 2 μg of ab176877 (red), or 2 μg of rabbit normal IgG Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730 (gray) and 20 μl of Protein A/G sepharose beads. The immunoprecipitated DNA was quantified by real time PCR (Taqman approach for active and inactive loci, Sybr green approach for heterochromatic loci).

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  ChIC/CUT&RUN sequencing - Anti-Histone H3 (mono methyl K4) antibody [ERP16597] - ChIP Grade (ab176877)

  CUT&RUN was performed using the ChIC/CUT&RUN pAG-MNAse ChIC/CUT&RUN pAG-MNase ab285373, 2.5 x 10^5 HeLa cells and 2μg of ab176877 [ERP16597]. The resulting DNA was sequenced on the NovaSeq 6000 (PE150).
  Additional screenshots of mapped reads can be downloaded here.

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  ChIP-sequencing - Anti-Histone H3 (mono methyl K4) antibody [ERP16597] - ChIP Grade (ab176877)

  Chromatin was prepared from HeLa cells. Cells were fixed with 1% formaldehyde for 10 minutes. ChIP was performed with 10^7 HeLa cells and 4 μg of ab176877 [ERP16597]. 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 (mono methyl K4) antibody [ERP16597] - ChIP Grade (ab176877)

  Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (Human epithelial cell line from cervix adenocarcinoma) cells labeling Histone H3 (mono methyl K4) with ab176877 at 1/1000 dilution, followed by goat anti-rabbit Alexa Fluor^® 488 (IgG) (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077) secondary antibody at 1/400 dilution (green). Nuclear staining on HeLa cell line is observed. The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291 (anti-Tubulin mouse mAb) at 1/500 dilution and Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed ab150120 (Alexa Fluor^® 594 goat anti-mouse secondary) at 1/500 dilution (red).
  The negative controls are as follows;
  1. ab176877 at 1/1000 dilution followed by Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed ab150120 (goat anti-mouse Alexa Fluor^® 594 secondary) at 1/500 dilution.
  2. Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291 (anti-Tubulin mouse mAb) at 1/500 dilution followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077 (goat anti-rabbit Alexa Fluor^® 488; IgG H&L) at 1/400 dilution.

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H3 (mono methyl K4) antibody [ERP16597] - ChIP Grade (ab176877)

  Immunohistochemical analysis of paraffin-embedded rat colon tissue labeling Histone H3 (mono methyl K4) with ab176877 at 1/1000 dilution, followed by prediluted Goat Anti-Rabbit IgG H&L (HRP). Nucleus staining on glandular epithelium of colon tissue is observed. Counterstained with hematoxylin.
  

  Negative control: PBS instead of primary ab, secondary ab is prediluted Goat Anti-Rabbit IgG H&L (HRP).

  Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

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  Western blot - Anti-Histone H3 (mono methyl K4) antibody [ERP16597] - ChIP Grade (ab176877)

  Blocking/Dilution buffer: 5% NFDM/TBST.

  All lanes: Western blot - Anti-Histone H3 (mono methyl K4) antibody [ERP16597] - ChIP Grade (ab176877) at 1/5000 dilution

  Lane 1: HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysates at 10 µg

  Lane 2: NIH/3T3 (Mouse embryo fibroblast cell line) whole cell lysates at 10 µg

  Secondary

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

  Predicted band size: 15 kDa

  Observed band size: 15 kDa

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  Peptide Array - Anti-Histone H3 (mono methyl K4) antibody [ERP16597] - ChIP Grade (ab176877)

  ab176877 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.

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H3 (mono methyl K4) antibody [ERP16597] - ChIP Grade (ab176877)

  Immunohistochemical analysis of paraffin-embedded human colon tissue labeling Histone H3 (mono methyl K4) with ab176877 at 1/1000 dilution, followed by prediluted Goat Anti-Rabbit IgG H&L (HRP). Nucleus staining on glandular epithelium of colon tissue is observed. Counterstained with hematoxylin.
  

  Negative control: PBS instead of primary ab, secondary ab is prediluted Goat Anti-Rabbit IgG H&L (HRP).

  Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• 

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H3 (mono methyl K4) antibody [ERP16597] - ChIP Grade (ab176877)

  Immunohistochemical analysis of paraffin-embedded mouse colon tissue labeling Histone H3 (mono methyl K4) with ab176877 at 1/1000 dilution, followed by prediluted Goat Anti-Rabbit IgG H&L (HRP). Nucleus staining on glandular epithelium of colon tissue is observed. Counterstained with hematoxylin.
  

  Negative control: PBS instead of primary ab, secondary ab is prediluted Goat Anti-Rabbit IgG H&L (HRP).

  Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• 

  ChIC/CUT&RUN sequencing - Anti-Histone H3 (mono methyl K4) antibody [ERP16597] - ChIP Grade (ab176877)

  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 2 µg of ab176877 [ERP16597]. 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.
  
  The ChIP data was conducted on chromatin prepared from HeLa cells. Cells were fixed with 1% formaldehyde for 10 minutes. ChIP was performed with 10^7 HeLa cells and 4 µg of ab176877. ChIP DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 30 million reads.
  
  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.