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 Anti-Histone H3 (tri methyl K27) antibody [EPR18607] - ChIP Grade (ab192985). 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.

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 25 µg of chromatin, 2 µg of ab192985 (blue), and 20 µl of Anti rabbit IgG sepharose beads. 2 μg of rabbit normal IgG was added to the beads control (yellow). The immunoprecipitated DNA was quantified by real time PCR (Sybr green approach).

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (Human epithelial cells from cervix adenocarcinoma) cells labeling Histone H3 (tri methyl K27) with ab192985 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG (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: ab192985 at 1/1000 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.

All lanes : Anti-Histone H3 (tri methyl K27) antibody [EPR18607] - ChIP Grade (ab192985) at 1/1000 dilution

Lane 1 : HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate prepared using 1% SDS hot lysis method
Lane 2 : HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate prepared using RIPA lysis method

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/5000 dilution

Predicted band size: 15 kDa
Observed band size: 15 kDa


Exposure time: 3 minutes

Blocking/Diluting buffer: 5% NFDM/TBST

For this product, we recommend 1% SDS hot lysis method.

For lysate preparation protocol, please refer to the protocol book in the protocol section or here (downloadable copy).

Immunohistochemical analysis of paraffin-embedded human colon tissue labeling Histone H3 (tri methyl K27) with ab192985 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Nuclear staining on 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) (ab97051) at 1/500 dilution.

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

All lanes : Anti-Histone H3 (tri methyl K27) antibody [EPR18607] - ChIP Grade (ab192985) at 1/1000 dilution

Lane 1 : HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysates with 5% NFDM/TBST
Lane 2 : HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysates with 2% BSA/TBST

Lysates/proteins at 15 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) (Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated)

Predicted band size: 15 kDa
Observed band size: 15 kDa


Exposure time: 40 seconds

We recommend to use 2% BSA as blocking and antibody dilution buffer.

Anti-Histone H3 (tri methyl K27) antibody [EPR18607] - ChIP Grade (ab192985) at 1/1000 dilution + NIH/3T3 (Mouse embryonic fibroblast cell line) whole cell lysate at 10 µg

Secondary
Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/50000 dilution

Predicted band size: 15 kDa
Observed band size: 15 kDa


Exposure time: 1 second

Blocking/Dilution buffer: 5% BSA/TBST

All lanes : Anti-Histone H3 (tri methyl K27) antibody [EPR18607] - ChIP Grade (ab192985) at 1/1000 dilution

Lane 1 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate
Lane 2 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate with H3K4Me1 peptide at 5 µg
Lane 3 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate with H3K4Me2 peptide at 5 µg
Lane 4 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate with H3K4Me3 peptide at 5 µg
Lane 5 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate with H3K9Me1 peptide at 5 µg
Lane 6 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate with H3K9Me2 peptide at 5 µg
Lane 7 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate with H3K9Me3 peptide at 5 µg
Lane 8 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate with H3K27Me1 peptide at 5 µg
Lane 9 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate with H3K27Me2 peptide at 5 µg
Lane 10 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate with H3K27Me3 peptide at 5 µg
Lane 11 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate with H3K27 unmodified peptide at 5 µg

Lysates/proteins at 10 µg per lane.

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

Developed using the ECL technique.

Predicted band size: 15 kDa


Exposure time: 5 seconds

The antibody is blocked by tri methyl K27 peptide (lane 10) and slightly by di methyl K27 peptide (lane 9, there is 14% cross reactivity with di methyl K27 as determined by ELISA).

All lanes : Anti-Histone H3 (tri methyl K27) antibody [EPR18607] - ChIP Grade (ab192985) at 1/1000 dilution

Lane 1 : HeLa (Human epithelial cell line from cervix adenocarcinoma)
Lane 2 : EED-/- mouse whole cell lysate
Lane 3 : Wild type mouse ES whole cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/50000 dilution

Developed using the ECL technique.

Predicted band size: 15 kDa


Exposure time: 8 minutes

Peptide array analysis was performed using ab192985 at a concentration of 0.1 µg/ml, followed by Goat Anti-Rabbit IgG, (H+L), Fluo 647nm conjugated secondary antibody at a 1/50,000 dilution.

ab192985 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, can be downloaded here.

ELISA analysis was performed on 1 µg/ml of antigen using ab192985 at a concentration range of 0-0.25 µg/ml, followed by Alkaline Phosphatase-conjugates AffiniPure Goat anti-rabbit IgG (H&L) secondary antibody at a 1/2,500 dilution.

All batches of ab192985 are tested in ELISA against peptides to different Histone H3 modifications. Results show strong binding to Histone H3 - tri methyl K27 immunizing peptide, indicating that this antibody specifically recognizes the Histone H3 - tri methyl K27 modification. Weak binding (14%) was also detected against H3 - di methyl K27 modification.

Immunohistochemical analysis of paraffin-embedded mouse colon tissue labeling Histone H3 (tri methyl K27) with ab192985 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Nuclear staining on mouse colon tissue 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.

Immunohistochemical analysis of paraffin-embedded rat kidney tissue labeling Histone H3 (tri methyl K27) with ab192985 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Nuclear staining on rat kidney tissue 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.