Anti-Histone H3 (unmodified K27) antibody [EPR23553-22]

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Histone H3 (unmodified K27) antibody [EPR23553-22] (AB308248)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized HeLa (human cervix adenocarcinoma epithelial cell) cells labelling Histone H3 (unmodified K27) with ab308248 at 1/200 (2.08 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 2ug/ml dilution (Green). Confocal image showing nuclear staining in HeLa cells.Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8). ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 2.5ug/ml dilution (Red). The Nuclear counterstain was DAPI (Blue). Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2ug/ml dilution.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H3 (unmodified K27) antibody [EPR23553-22] (AB308248)

Immunohistochemical analysis of paraffin-embedded Human gastric adenoc tissue labeling Histone H3 (unmodified K27) with ab308248 at 1/4000 (0.104 ug/ml) followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP polymer). Nuclear staining on human gastric adenocarcinoma. The section was incubated with ab308248 at 4 ℃ overnight. Counterstained with Hematoxylin. Secondary antibody only control : Secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP polymer). Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0)

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H3 (unmodified K27) antibody [EPR23553-22] (AB308248)

Immunohistochemical analysis of paraffin-embedded Human lung tissue labeling Histone H3 (unmodified K27) with ab308248 at 1/4000 (0.104 ug/ml) followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP polymer). Nuclear staining on human lung. The section was incubated with ab308248 at 4 ℃ overnight. Counterstained with Hematoxylin. Secondary antibody only control : Secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP polymer). Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0)

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Histone H3 (unmodified K27) antibody [EPR23553-22] (AB308248)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized HeLa (human cervical adenocarcinoma epithelial cell) cells labelling Histone H3 (unmodified K27) with ab308248 at 1/500 dilution (0.1 ug)/Red (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/5000 dilution was used as the secondary antibody.

• ChIP-seq

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ChIP-sequencing - Anti-Histone H3 (unmodified K27) antibody [EPR23553-22] (AB308248)

Chromatin was prepared from HeLa cells. Cells were fixed with 1% formaldehyde for 10 minutes. ChIP was performed with 10^7 cells and 4 µg of Histone H3 (unmodified K27) (ab308248). ChIP DNA was sequenced on the Illumina NovaSeq6000 to a depth of 30 million reads. Line 1 shows the input as the negative control and line 2 shows results of H3 (unmodified K27) (ab308248). H3 acetyl K27 (ab177178) and H3 tri Methyl K27(ab192985) are shown as controls in lines 3 and 4 respectively. The reads using the unmodified H3K27 antibody (ab308248) do not align with the peaks from the experiment with antibodies to modifications (ab177178 and ab192985), which is as expected for this target.

• ChIP-seq

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ChIP-sequencing - Anti-Histone H3 (unmodified K27) antibody [EPR23553-22] (AB308248)

Chromatin was prepared from HeLa cells. Cells were fixed with 1% formaldehyde for 10 minutes. ChIP was performed with 10^7 cells and 4 µg of Histone H3 (unmodified K27) (ab308248). ChIP DNA was sequenced on the Illumina NovaSeq6000 to a depth of 30 million reads. Line 1 shows the input as the negative control and line 2 shows results of H3 (unmodified K27) (ab308248). H3 acetyl K27 (ab177178) and H3 tri Methyl K27(ab192985) are shown as controls in lines 3 and 4 respectively. The reads using the unmodified H3K27 antibody (ab308248) do not align with the peaks from the experiment with antibodies to modifications (ab177178 and ab192985), which is as expected for this target.

• ChIP-seq

Supplier Data

ChIP-sequencing - Anti-Histone H3 (unmodified K27) antibody [EPR23553-22] (AB308248)

Chromatin was prepared from HeLa cells. Cells were fixed with 1% formaldehyde for 10 minutes. ChIP was performed with 10^7 cells and 4 µg of Histone H3 (unmodified K27) (ab308248). ChIP DNA was sequenced on the Illumina NovaSeq6000 to a depth of 30 million reads. Line 1 shows the input as the negative control and line 2 shows results of H3 (unmodified K27) (ab308248). H3 acetyl K27 (ab177178) and H3 tri Methyl K27(ab192985) are shown as controls in lines 3 and 4 respectively. The reads using the unmodified H3K27 antibody (ab308248) do not align with the peaks from the experiment with antibodies to modifications (ab177178 and ab192985), which is as expected for this target.

• ChIP-seq

Supplier Data

ChIP-sequencing - Anti-Histone H3 (unmodified K27) antibody [EPR23553-22] (AB308248)

Chromatin was prepared from HeLa cells. Cells were fixed with 1% formaldehyde for 10 minutes. ChIP was performed with 10^7 cells and 4 µg of Histone H3 (unmodified K27) (ab308248). ChIP DNA was sequenced on the Illumina NovaSeq6000 to a depth of 30 million reads. Line 1 shows the input as the negative control and line 2 shows results of H3 (unmodified K27) (ab308248). H3 acetyl K27 (ab177178) and H3 tri Methyl K27(ab192985) are shown as controls in lines 3 and 4 respectively. The reads using the unmodified H3K27 antibody (ab308248) do not align with the peaks from the experiment with antibodies to modifications (ab177178 and ab192985), which is as expected for this target.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H3 (unmodified K27) antibody [EPR23553-22] (AB308248)

Immunohistochemical analysis of paraffin-embedded Rat lung tissue labeling Histone H3 (unmodified K27) with ab308248 at 1/4000 (0.104 ug/ml) followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP polymer). Nuclear staining on rat lung. The section was incubated with ab308248 at 4 ℃ overnight. Counterstained with Hematoxylin. Secondary antibody only control : Secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP polymer). Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0)

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Histone H3 (unmodified K27) antibody [EPR23553-22] (AB308248)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized NIH/3T3 (mouse embryonic fibroblast) cells labelling Histone H3 (unmodified K27) with ab308248 at 1/500 dilution (0.1 ug)/Red (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/5000 dilution was used as the secondary antibody.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Histone H3 (unmodified K27) antibody [EPR23553-22] (AB308248)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized NIH/3T3 (mouse embryonic fibroblast) cells labelling Histone H3 (unmodified K27) with ab308248 at 1/200 (2.08 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 2ug/ml dilution (Green). Confocal image showing nuclear staining in NIH/3T3 cells.Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8). ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 2.5ug/ml dilution (Red). The Nuclear counterstain was DAPI (Blue). Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2ug/ml dilution.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H3 (unmodified K27) antibody [EPR23553-22] (AB308248)

Immunohistochemical analysis of paraffin-embedded Mouse lung tissue labeling Histone H3 (unmodified K27) with ab308248 at 1/4000 (0.104 ug/ml) followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP polymer). Nuclear staining on mouse lung. The section was incubated with ab308248 at 4 ℃ overnight. Counterstained with Hematoxylin. Secondary antibody only control : Secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP polymer). Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0)

• WB

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Western blot - Anti-Histone H3 (unmodified K27) antibody [EPR23553-22] (AB308248)

Blocking and diluting buffer and concentration : 1% BSA/TBST The lysates of lane2 and lane3 were freshly made and used for Western blotting immediately to minimize protein degradation. Exposure time : Lane 1-2 : 26 seconds, lane 3 : 15 seconds

All lanes:

Western blot - Anti-Histone H3 (unmodified K27) antibody [EPR23553-22] (ab308248) at 1/1000 dilution

Lane 1:

Human spleen tissue lysate at 20 µg

Lane 2:

Mouse spleen tissue lysate at 20 µg

Lane 3:

Rat spleen tissue lysate at 20 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

Observed band size: 15 kDa

false

• WB

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Western blot - Anti-Histone H3 (unmodified K27) antibody [EPR23553-22] (AB308248)

Blocking and diluting buffer and concentration : 1% BSA/TBST Lysates were freshly made and used for Western blotting immediately to minimize protein degradation. Exposure time : Lane 1-3 : 26 seconds, lane 4-7 : 15 seconds

All lanes:

Western blot - Anti-Histone H3 (unmodified K27) antibody [EPR23553-22] (ab308248) at 1/1000 dilution

Lane 1:

HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

NIH/3T3 (mouse embryonic fibroblast) whole cell lysate at 20 µg

Lane 3:

PC-12 (rat adrenal gland pheochromocytoma cell) whole cell lysate at 20 µg

Lane 4:

THP-1 (human monocytic leukemia monocyte) whole cell lysate at 20 µg

Lane 5:

Jurkat (human T cell leukemia T lymphocyte) whole cell lysate at 20 µg

Lane 6:

RAW 264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysate at 20 µg

Lane 7:

2.4G2 (rat B cell lymphoma B lymphocyte) whole cell lysate at 20 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

Observed band size: 15 kDa

false

• WB

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Western blot - Anti-Histone H3 (unmodified K27) antibody [EPR23553-22] (AB308248)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

This antibody does not cross-react with human H2A, H2B and H4C1.

This blot was developed using a high sensitivity ECL substrate. The high-sensitivity ECL substrate used allows for the detection of proteins in the mid-femtogram range.

In Western blot, anti-His antibody (ab213204) staining at 1/5000 dilution.

All lanes:

Western blot - Anti-Histone H3 (unmodified K27) antibody [EPR23553-22] (ab308248) at 1/1000 dilution

Lane 1:

His tagged Human H2A recombinant protein at 100 ng

Lane 2:

His tagged Human H2B recombinant protein at 100 ng

Lane 3:

His tagged Human H3C1 recombinant protein at 50 ng

Lane 4:

His tagged Human H4C1 recombinant protein at 200 ng

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

Observed band size: 15 kDa

true

Exposure time: 180s

• PepArr

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Peptide Array - Anti-Histone H3 (unmodified K27) antibody [EPR23553-22] (AB308248)

Peptide array analysis of ab308248 at (0.1ug/ml) followed by a Goat Anti-Rabbit IgG, (H+L), Fluo 647nm conjugated at 1 : 50,000 dilution. ab308248 has been 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 the area under the 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 a full list of all peptides and information on the position of each peptide in the diagram, can be downloaded here.

• PepArr

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Peptide Array - Anti-Histone H3 (unmodified K27) antibody [EPR23553-22] (AB308248)

Peptide array analysis of ab308248 at (0.1ug/ml) followed by a Goat Anti-Rabbit IgG, (H+L), Fluo 647nm conjugated at 1 : 50,000 dilution. The affinity of ab308248 to the un-modified Histone H3 K27 sequence and peptides modified at K27 from our peptide array are shown here as a histogram. AUC stands for Area under Curve.