Anti-Histone H3 (di methyl K4) antibody [EPR17707] - ChIP Grade

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H3 (di methyl K4) antibody [EPR17707] - ChIP Grade (AB176878)

Immunohistochemical analysis of paraffin-embedded human colon tissue labeling Histone H3 (di methyl K4) with ab176878 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Nuclear staining on human colon is observed. Counterstained with hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is 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.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Histone H3 (di methyl K4) antibody [EPR17707] - ChIP Grade (AB176878)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (Human epithelial cell line from cervix adenocarcinoma) cells labeling Histone H3 (di methyl K4) with ab176878 at 1/500 dilution, followed by 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 counterstain is DAPI (blue).

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

The negative controls are as follows : -

-ve control 1 : ab176878 at 1/500 dilution, followed by Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) (ab150120) secondary antibody at 1/1000 dilution.

-ve control 2 : Anti-alpha Tubulin mouse MAb (ab7291) at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.

• ChIP

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ChIP - Anti-Histone H3 (di methyl K4) antibody [EPR17707] - ChIP Grade (AB176878)

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 minutes. The ChIP was performed with 25μg of chromatin, 2μg of ab176878 (blue), and 20μl of Anti Rabbit IgG sepharose beads. 2μg of rabbit normal IgG was added to the beads control (yellow). 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.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H3 (di methyl K4) antibody [EPR17707] - ChIP Grade (AB176878)

Immunohistochemical analysis of paraffin-embedded mouse colon tissue labeling Histone H3 (di methyl K4) with ab176878 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Nuclear staining on mouse colon is observed. Counterstained with hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is 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.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-Histone H3 (di methyl K4) antibody [EPR17707] - ChIP Grade (AB176878)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized NIH/3T3 (Mouse embryonic fibroblast cell line) cells labeling Histone H3 (di methyl K4) with ab176878 at 1/500 dilution, followed by 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 counterstain is DAPI (blue).

Tubulin is detected with Anti-alpha Tubulin mouse MAb (ab7291) at 1/1000 dilution followed by Goat Anti-Mouse IgG H&L (AlexaFluor^®594) (ab150120) secondary antibody at 1/1000 dilution (red)

The negative controls are as follows : -

-ve control 1 : ab176878 at 1/500 dilution, followed by Goat Anti-Mouse IgG H&L (AlexaFluor^®594) (ab150120) secondary antibody at 1/1000 dilution.

-ve control 2 : Anti-alpha Tubulin mouse MAb (ab7291) at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H3 (di methyl K4) antibody [EPR17707] - ChIP Grade (AB176878)

Immunohistochemical analysis of paraffin-embedded rat colon tissue labeling Histone H3 (di methyl K4) with ab176878 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Nuclear staining on rat colon is observed. Counterstained with hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is 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.

• WB

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Western blot - Anti-Histone H3 (di methyl K4) antibody [EPR17707] - ChIP Grade (AB176878)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-Histone H3 (di methyl K4) antibody [EPR17707] - ChIP Grade (ab176878) 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

Lane 3:

PC-12 (Rat adrenal gland pheochromocytoma cell line) whole cell lysate at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 15 kDa

Observed band size: 15 kDa

false

Exposure time: 10s

• ChIC/CUT&RUN-seq

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ChIC/CUT&RUN sequencing - Anti-Histone H3 (di methyl K4) antibody [EPR17707] - ChIP Grade (AB176878)

ChIC/CUT&RUN was performed using a pAG-MNAse at a final concentration of 700 ng/mL, 2 x 10^5 HeLa (Human cervix adenocarcinoma epithelial cell line) cells and 2 µg of ab176878 [EPR17707]. The resulting DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 10 million reads. The negative IgG 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 ab176878. 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 Product Protocol section. The University of Geneva owns patents relevant to ChIC (Chromatin Immuno-Cleavage) methods.

• ChIC/CUT&RUN-seq

Supplier Data

ChIC/CUT&RUN sequencing - Anti-Histone H3 (di methyl K4) antibody [EPR17707] - ChIP Grade (AB176878)

CUT&RUN was performed using the ChIC/CUT&RUN pAG-MNAse ab285373, 105 HeLa cells and 2μg of ab176878 [EPR17707]. The resulting DNA was sequenced on the NovaSeq 6000 (PE150). Additional screenshots of mapped reads can be downloaded here.

• PepArr

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Peptide Array - Anti-Histone H3 (di methyl K4) antibody [EPR17707] - ChIP Grade (AB176878)

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 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 Product Protocol section.