Anti-Histone H3 (tri methyl K27) 抗体 [EPR18607] - ChIP Grade

• ChIP-seq

Supplier Data

ChIP-sequencing - Anti-Histone H3 (tri methyl K27) antibody [EPR18607] - ChIP Grade (AB192985)

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.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Histone H3 (tri methyl K27) antibody [EPR18607] - ChIP Grade (AB192985)

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.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H3 (tri methyl K27) antibody [EPR18607] - ChIP Grade (AB192985)

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.

• ChIP

Supplier Data

ChIP - Anti-Histone H3 (tri methyl K27) antibody [EPR18607] - ChIP Grade (AB192985)

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).

• ChIP-seq

AbReview81542****

ChIP-sequencing - Anti-Histone H3 (tri methyl K27) antibody [EPR18607] - ChIP Grade (AB192985)

Chromatin was prepared from human Nuclear cell lysate K562 cells. Cells were fixed with 1% formaldehyde for 10 minutes. Cells were incubated with the primary antibody with PBS + 0.1% Tween-20 for 1 hour at 25°C

This image is courtesy of an Abreview submitted by Andrew Perez

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H3 (tri methyl K27) antibody [EPR18607] - ChIP Grade (AB192985)

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.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H3 (tri methyl K27) antibody [EPR18607] - ChIP Grade (AB192985)

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.

• WB

Lab

Western blot - Anti-Histone H3 (tri methyl K27) antibody [EPR18607] - ChIP Grade (AB192985)

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 section of the website and/or here.

All lanes:

Western blot - 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 at 20 µg

Lane 2:

HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate prepared using RIPA lysis method at 20 µg

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

false

Exposure time: 3min

• WB

Supplier Data

Western blot - Anti-Histone H3 (tri methyl K27) antibody [EPR18607] - ChIP Grade (AB192985)

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:

Western blot - 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 at 10 µg

Lane 2:

HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 10 µg with H3K4Me1 peptide

Lane 3:

HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 10 µg with H3K4Me2 peptide

Lane 4:

HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 10 µg with H3K4Me3 peptide

Lane 5:

HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 10 µg with H3K9Me1 peptide

Lane 6:

HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 10 µg with H3K9Me2 peptide

Lane 7:

HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 10 µg with H3K9Me3 peptide

Lane 8:

HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 10 µg with H3K27Me1 peptide

Lane 9:

HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 10 µg with H3K27Me2 peptide

Lane 10:

HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 10 µg with H3K27Me3 peptide

Lane 11:

HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 10 µg with H3K27 unmodified peptide

Secondary

All lanes:

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

Observed band size: 15 kDa

true

Exposure time: 5s

• WB

Lab

Western blot - Anti-Histone H3 (tri methyl K27) antibody [EPR18607] - ChIP Grade (AB192985)

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

All lanes:

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

All lanes:

HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysates at 15 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 15 kDa

Observed band size: 15 kDa

false

Exposure time: 40s

• WB

Supplier Data

Western blot - Anti-Histone H3 (tri methyl K27) antibody [EPR18607] - ChIP Grade (AB192985)

Blocking/Dilution buffer : 5% BSA/TBST

All lanes:

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

All lanes:

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) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/50000 dilution

Predicted band size: 15 kDa,45 kDa

Observed band size: 15 kDa,30 kDa

false

Exposure time: 1s

• ELISA

Supplier Data

ELISA - Anti-Histone H3 (tri methyl K27) antibody [EPR18607] - ChIP Grade (AB192985)

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.

• WB

Supplier Data

Western blot - Anti-Histone H3 (tri methyl K27) antibody [EPR18607] - ChIP Grade (AB192985)

All lanes:

Western blot - 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) at 10 µg

Lane 2:

EED-/- mouse whole cell lysate at 10 µg

Lane 3:

Wild type mouse ES whole cell lysate at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/50000 dilution

Predicted band size: 15 kDa

true

Exposure time: 8min

• PepArr

Supplier Data

Peptide Array - Anti-Histone H3 (tri methyl K27) antibody [EPR18607] - ChIP Grade (AB192985)

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, is available under the Product Protocol section.