Anti-Histone H3 (tri methyl K9) antibody [EPR16601] - ChIP Grade

16 product images

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  ChIC/CUT&RUN sequencing - Anti-Histone H3 (tri methyl K9) antibody [EPR16601] - ChIP Grade (ab176916)

  CUT&RUN was performed using the ChIC/CUT&RUN pAG-MNAse ChIC/CUT&RUN pAG-MNase ab285373, 105 HeLa cells and 2μg of ab176916 [EPR16601]. The resulting DNA was sequenced on the NovaSeq 6000 (PE150).
  Additional screenshots of mapped reads can be downloaded here.

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  ChIP - Anti-Histone H3 (tri methyl K9) antibody [EPR16601] - ChIP Grade (ab176916)

  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 ab176916 (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.

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  This experiment and image is courtesy of Dr Marek Bartosovic, Gonçalo Castelo-Branco Group, Karolinska Institutet.

  CUT&Tag sequencing - Anti-Histone H3 (tri methyl K9) antibody [EPR16601] - ChIP Grade (ab176916)

  CUT&Tag-seq was performed using 200,000 Oli-neu (Oligodendrocyte progenitor) cells. Cells were permeabilized with 0.05% Digitonin and 0.01% NP-40 for 3 minutes. A 1:100 dilution of Recombinant Anti-Histone H3 (tri methyl K9) antibody [EPR16601] - ChIP Grade (ab176916) was used, along with a Guinea pig anti-rabbit Secondary. DNA was seg using Illumina NovaSeq S Prime to a depth of 19 million reads.
  

  This image is courtesy of Dr Marek Bartosovic, Gonçalo Castelo-Branco Group, Karolinska Institutet.

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  ChIP-sequencing - Anti-Histone H3 (tri methyl K9) antibody [EPR16601] - ChIP Grade (ab176916)

  Chromatin was prepared from HeLa cells. Cells were fixed with 1% formaldehyde for 10 minutes. ChIP was performed with 107 HeLa cells and 4 μg of ab176916 [EPR16601]. 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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  Western blot - Anti-Histone H3 (tri methyl K9) antibody [EPR16601] - ChIP Grade (ab176916)

  Blocking/Dilution buffer: 5% NFDM/TBST.

  Exposure time: Lane 1: 1 second; Lane 2: 4 seconds.

  All lanes: Western blot - Anti-Histone H3 (tri methyl K9) antibody [EPR16601] - ChIP Grade (ab176916) at 1/1000 dilution

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

  Lane 2: NIH/3T3 (Mouse embryonic fibroblast cell line) whole cell lysate at 10 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: 15 kDa

  Observed band size: 15 kDa

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H3 (tri methyl K9) antibody [EPR16601] - ChIP Grade (ab176916)

  Immunohistochemical analysis of paraffin-embedded human colon tissue labeling Histone H3 (tri methyl K9) with ab176916 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/500 dilution. Nuclear staining on human colon 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) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/500 dilution.

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

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  Immunocytochemistry/ Immunofluorescence - Anti-Histone H3 (tri methyl K9) antibody [EPR16601] - ChIP Grade (ab176916)

  Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized NIH/3T3 (Mouse embryonic fibroblast cell line) cells labeling Histone H3 (tri methyl K9) with ab176916 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing nuclear staining on NIH/3T3 cell line. The nuclear counter stain is DAPI (blue).

  Tubulin is detected with Anti-alpha Tubulin mouse MAb (Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291) at 1/1000 dilution, followed by Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) (Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed ab150120) secondary antibody at 1/1000 dilution (red).

  The negative controls are as follows:
  -ve control 1: ab176916 at 1/2000 dilution followed by Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) (Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed ab150120) secondary antibody at 1/1000 dilution.
  -ve control 2: Anti-alpha Tubulin mouse MAb (Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291) at 1/1000 dilution followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077) secondary antibody at 1/1000 dilution.

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  Dot Blot - Anti-Histone H3 (tri methyl K9) antibody [EPR16601] - ChIP Grade (ab176916)

  Dot blot analysis of Histone H3 (tri methyl K9) peptide (Lane 1), Histone H3K9 unmodified peptide (Lane 2), Histone H3 (crotonyl K4) peptide (Lane 3) and Histone H3K4 unmodified peptide (Lane 4) labeled using ab176916 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) secondary antibody at 1/1000 dilution.

  Blocking/Dilution buffer: 5% NFDM/TBST.

  Exposure time: 3 minutes.

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  Peptide Array - Anti-Histone H3 (tri methyl K9) antibody [EPR16601] - ChIP Grade (ab176916)

  ab176916 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 (tri methyl K9) antibody [EPR16601] - ChIP Grade (ab176916)

  Immunohistochemical analysis of paraffin-embedded mouse liver tissue labeling Histone H3 (tri methyl K9) with ab176916 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/500 dilution. Nuclear staining on mouse liver 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) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/500 dilution.

  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 (tri methyl K9) antibody [EPR16601] - ChIP Grade (ab176916)

  Immunohistochemical analysis of paraffin-embedded rat liver tissue labeling Histone H3 (tri methyl K9) with ab176916 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/500 dilution. Nuclear staining on rat liver 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) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/500 dilution.

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

• 

  Immunocytochemistry/ Immunofluorescence - Anti-Histone H3 (tri methyl K9) antibody [EPR16601] - ChIP Grade (ab176916)

  Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (Human epithelial cell line from cervix adenocarcinoma) cells labeling Histone H3 (tri methyl K9) with ab176916 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing nuclear staining on HeLa cell line. The nuclear counter stain is DAPI (blue).

  Tubulin is detected with Anti-alpha Tubulin mouse MAb (Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291) at 1/1000 dilution, followed by Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) (Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed ab150120) secondary antibody at 1/1000 dilution (red).

  The negative controls are as follows:
  -ve control 1: ab176916 at 1/2000 dilution followed by Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) (Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed ab150120) secondary antibody at 1/1000 dilution.
  -ve control 2: Anti-alpha Tubulin mouse MAb (Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291) at 1/1000 dilution followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077) secondary antibody at 1/1000 dilution.

• 

  ChIC/CUT&RUN sequencing - Anti-Histone H3 (tri methyl K9) antibody [EPR16601] - ChIP Grade (ab176916)

  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 ab176916 [EPR16601]. 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 ab176916. ChIP DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 30 million reads. The University of Geneva owns patents relevant to ChIC (Chromatin Immuno-Cleavage) methods.
  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.

• 

  This experiment and image is courtesy of Dr Marek Bartosovic, Gonçalo Castelo-Branco Group, Karolinska Institutet.

  CUT&Tag - Anti-Histone H3 (tri methyl K9) antibody [EPR16601] - ChIP Grade (ab176916)

  CUT&Tag-seq was performed using 200,000 Oli-neu (Oligodendrocyte progenitor) cells. Cells were permeabilized with 0.05% Digitonin and 0.01% NP-40 for 3 minutes. A 1:100 dilution of Recombinant Anti-Histone H3 (tri methyl K9) antibody [EPR16601] - ChIP Grade (ab176916) was used, along with a Guinea pig anti-rabbit Secondary. DNA was seg using Illumina NovaSeq S Prime to a depth of 19 million reads.
  This image is courtesy of Dr Marek Bartosovic, Gonçalo Castelo-Branco Group, Karolinska Institutet.
  The University of Geneva owns patents relevant to ChIC (Chromatin Immuno-Cleavage) methods.

• 

  Immunohistochemistry - Anti-Histone H3 (tri methyl K9) antibody [EPR16601] - ChIP Grade (ab176916)

  Immunohistochemical analysis of formalin fixed paraffin embedded human liver labelling Histone H3 (tri methyl K9) with ab176916 at a concentration of 0.07 µg/ml. The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with a OptiView DAB IHC Detection Kit. Heat mediated antigen retrieval was performed with DISCOVERY cell conditioning solution (CC1) 100°C, pH8.5 for 32mins.

  ab176916 Anti-Histone H3 (tri methyl K9) antibody [EPR16601] was incubated for 16mins at 37°C. Sections were counterstained with Hematoxylin II. Image inset shows absence of staining in secondary antibody only control.

  Customers are encouraged to optimise antigen retrieval conditions, antibody concentration, incubation times and temperature for best results in their own IHC assay workflow (automated and manual).

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  ChIP-sequencing - Anti-Histone H3 (tri methyl K9) antibody [EPR16601] - ChIP Grade (ab176916)

  Chromatin was prepared from Mouse Embryonic Fibroblasts cells. Cells were fixed with 1% formaldehyde for 10 minutes. ChIP was performed with 10^7 cells and 4 µg of Anti-Histone H3 (tri methyl K9) antibody (ab176916). ChIP DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 30 million reads. The Input control is also shown.
  
  Additional screenshots of mapped reads can be found in the Protocol booklet in the Support and downloads section.