Anti-Histone H3 antibody [1B1B2] - BSA and Azide free

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-Histone H3 antibody [1B1B2] - BSA and Azide free (AB325978)

This data was developed using ab309551, the same antibody clone in a different buffer formulation.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed 100% Methanol permeabilized HeLa (human cervical adenocarcinoma epithelial cell) cells labelling Histone H3 with ab309551 at 1/50 (20.52 ug/ml) dilution followed by ab150117 Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 dilution (Green). Confocal image showing nuclear staining in HeLa cell line. Image was taken with a confocal microscope(Leica-Microsystems TCS SP8). ab206369 Anti-beta Tubulin rabbit monoclonal antibody (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 dilution (Red). The Nuclear counterstain was DAPI (Blue). antibody only control : Secondary antibody is ab150117 Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 dilution.

• ChIP

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ChIP - Anti-Histone H3 antibody [1B1B2] - BSA and Azide free (AB325978)

This data was developed using ab309551, the same antibody clone in a different buffer formulation.

Chromatin was prepared from HeLa cells according to the Abcam X-ChIP protocol*. Cells were fixed with formaldehyde for 10min.The ChIP was performed with 25 µg of chromatin 2 µg of ab309551(red) or 2 µg of mouse IgG3 (gray) and 25 µl of Protein A/G Dynabeads. 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.*http : //www.abcam.com/resources?keywords=X%20ChIP%20protocol

• IP

Supplier Data

Immunoprecipitation - Anti-Histone H3 antibody [1B1B2] - BSA and Azide free (AB325978)

This data was developed using ab309551, the same antibody clone in a different buffer formulation.

Histone H3 was immunoprecipitated from 0.35 mg HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate with ab309551 at 1/50 dilution (2ug in 0.4mg lysates). Western blot was performed on the immunoprecipitate using ab309551 at 1/1000 dilution. mouse IgG for IP (HRP) (ab131368) was used at 1/5000 dilution. Lane 1 : HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate Lane 2 : ab309551 IP in HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate Lane 3 : Mouse IgG1 monoclonal isotype control (ab18443) instead of ab309551 in HeLa whole cell lysate Blocking and dilution buffer and concentration : 5% NFDM/TBST. Exposure time : 32 seconds

All lanes:

Immunoprecipitation - Anti-Histone H3 antibody [1B1B2] - Nuclear Marker (<a href='/en-us/products/primary-antibodies/histone-h3-antibody-1b1b2-nuclear-marker-ab309551'>ab309551</a>) at 1/50 dilution

All lanes:

HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate

Secondary

All lanes:

Immunoprecipitation - Anti-mouse IgG for IP (HRP) (<a href='/en-us/products/secondary-antibodies/mouse-igg-for-ip-hrp-ab131368'>ab131368</a>) at 1/5000 dilution

false

Exposure time: 32s

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-Histone H3 antibody [1B1B2] - BSA and Azide free (AB325978)

This data was developed using ab309551, the same antibody clone in a different buffer formulation.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed 100% Methanol permeabilized NIH/3T3 (mouse embryonic fibroblast) cells labelling Histone H3 with ab309551 at 1/50 (20.52 ug/ml) dilution followed by ab150117 Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 dilution (Green). Confocal image showing nuclear staining in NIH/3T3 cell line. Image was taken with a confocal microscope(Leica-Microsystems TCS SP8). ab206369 Anti-beta Tubulin rabbit monoclonal antibody (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 dilution (Red). The Nuclear counterstain was DAPI (Blue). antibody only control : Secondary antibody is ab150117 Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 dilution.

• IP

Supplier Data

Immunoprecipitation - Anti-Histone H3 antibody [1B1B2] - BSA and Azide free (AB325978)

This data was developed using ab309551, the same antibody clone in a different buffer formulation.

Histone H3 was immunoprecipitated from 0.35 mg NIH/3T3 (mouse embryonic fibroblast) whole cell lysate with ab309551 at 1/50 dilution (2ug in 0.4mg lysates). Western blot was performed on the immunoprecipitate using ab309551 at 1/1000 dilution. mouse IgG for IP (HRP) (ab131368) was used at 1/5000 dilution. Lane 1 : NIH/3T3 (mouse embryonic fibroblast) whole cell lysate Lane 2 : ab309551 IP in NIH/3T3 (mouse embryonic fibroblast) whole cell lysate Lane 3 : Mouse IgG1 monoclonal isotype control (ab18443) instead of ab309551 in NIH/3T3 whole cell lysate Blocking and dilution buffer and concentration : 5% NFDM/TBST. Exposure time : 32 seconds

All lanes:

Immunoprecipitation - Anti-Histone H3 antibody [1B1B2] - Nuclear Marker (<a href='/en-us/products/primary-antibodies/histone-h3-antibody-1b1b2-nuclear-marker-ab309551'>ab309551</a>) at 1/50 dilution

All lanes:

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

Secondary

All lanes:

Immunoprecipitation - Anti-mouse IgG for IP (HRP) (<a href='/en-us/products/secondary-antibodies/mouse-igg-for-ip-hrp-ab131368'>ab131368</a>) at 1/5000 dilution

false

Exposure time: 32s

• IP

Supplier Data

Immunoprecipitation - Anti-Histone H3 antibody [1B1B2] - BSA and Azide free (AB325978)

This data was developed using ab309551, the same antibody clone in a different buffer formulation.

Histone H3 was immunoprecipitated from 0.35 mg C6 (rat glial tumor glial cell) whole cell lysate with ab309551 at 1/50 dilution (2ug in 0.4mg lysates). Western blot was performed on the immunoprecipitate using ab309551 at 1/1000 dilution. mouse IgG for IP (HRP) (ab131368) was used at 1/5000 dilution. Lane 1 : C6 (rat glial tumor glial cell) whole cell lysate Lane 2 : ab309551 IP in C6 (rat glial tumor glial cell) whole cell lysate Lane 3 : Mouse IgG1 monoclonal isotype control (ab18443) instead of ab309551 in C6 whole cell lysate Blocking and dilution buffer and concentration : 5% NFDM/TBST. Exposure time : 67 seconds

All lanes:

Immunoprecipitation - Anti-Histone H3 antibody [1B1B2] - Nuclear Marker (<a href='/en-us/products/primary-antibodies/histone-h3-antibody-1b1b2-nuclear-marker-ab309551'>ab309551</a>) at 1/50 dilution

All lanes:

C6 (rat glial tumor glial cell) whole cell lysate at 10 µg

Secondary

All lanes:

Immunoprecipitation - Anti-mouse IgG for IP (HRP) (<a href='/en-us/products/secondary-antibodies/mouse-igg-for-ip-hrp-ab131368'>ab131368</a>) at 1/5000 dilution

false

Exposure time: 67s

• WB

Supplier Data

Western blot - Anti-Histone H3 antibody [1B1B2] - BSA and Azide free (AB325978)

This data was developed using ab309551, the same antibody clone in a different buffer formulation.

Blocking and diluting buffer and concentration : 5% NFDM/TBST Exposure time : 4 seconds

All lanes:

Western blot - Anti-Histone H3 antibody [1B1B2] - Nuclear Marker (<a href='/en-us/products/primary-antibodies/histone-h3-antibody-1b1b2-nuclear-marker-ab309551'>ab309551</a>) at 1/1000 dilution

Lane 1:

MCF7 (human breast adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

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

Lane 3:

C6 (rat glial tumor glial cell) whole cell lysate at 20 µg

Lane 4:

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

Secondary

All lanes:

Peroxidase-Conjugated Goat anti-Mouse IgG (H+L) at 1/10000 dilution

Observed band size: 18 kDa

false

Exposure time: 4s

• WB

Supplier Data

Western blot - Anti-Histone H3 antibody [1B1B2] - BSA and Azide free (AB325978)

This data was developed using ab309551, the same antibody clone in a different buffer formulation.

Blocking and diluting buffer and concentration : 5% NFDM/TBST Exposure time : Lane 1 : 32 seconds; Lane 2 : 93 seconds

All lanes:

Western blot - Anti-Histone H3 antibody [1B1B2] - Nuclear Marker (<a href='/en-us/products/primary-antibodies/histone-h3-antibody-1b1b2-nuclear-marker-ab309551'>ab309551</a>) at 1/1000 dilution

Lane 1:

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

Lane 2:

MCF7 (human breast adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Anti-mouse IgG for IP (HRP) (<a href='/en-us/products/secondary-antibodies/mouse-igg-for-ip-hrp-ab131368'>ab131368</a>) at 1/5000 dilution

Observed band size: 18 kDa

false

• Dot

Supplier Data

Dot Blot - Anti-Histone H3 antibody [1B1B2] - BSA and Azide free (AB325978)

This data was developed using ab309551, the same antibody clone in a different buffer formulation.

Dot blot analysis of Histone H3 using ab309551 at 1000 followed by a Peroxidase-Conjugated Goat anti-Mouse IgG (H+L) at 10000 dilution. Lane 1 : Human Histone H3 peptide Lane 2 : Human Histone H3 (phospho T118) peptide Exposure time : 59 seconds Blocking and diluting buffer and concentration : 5% NFDM/TBST

• WB

Supplier Data

Western blot - Anti-Histone H3 antibody [1B1B2] - BSA and Azide free (AB325978)

This data was developed using ab309551, the same antibody clone in a different buffer formulation.

Blocking and diluting buffer and concentration : 5% NFDM/TBST. Exposure time : 4 seconds

All lanes:

Western blot - Anti-Histone H3 antibody [1B1B2] - Nuclear Marker (<a href='/en-us/products/primary-antibodies/histone-h3-antibody-1b1b2-nuclear-marker-ab309551'>ab309551</a>) at 1/1000 dilution

Lane 1:

RAW 264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysate at 10 µg

Lane 2:

NIH/3T3 (mouse embryonic fibroblast) whole cell lysate at 10 µg

Secondary

All lanes:

Peroxidase-Conjugated Goat anti-Mouse IgG (H+L) at 1/10000 dilution

Observed band size: 18 kDa

false

Exposure time: 4s

• PepArr

Supplier Data

Peptide Array - Anti-Histone H3 antibody [1B1B2] - BSA and Azide free (AB325978)

This data was developed using ab309551, the same antibody clone in a different buffer formulation.

Peptide array analysis of ab309551 at 0.2µg/ml followed by a Goat Anti-Mouse IgG (H+L) Fluo 647nm conjugated at 50000 dilution. ab309551 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 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. ab309551 shows weak cross-reactivity with H3 T118p in PA but the Dot blot shows specific.