Anti-STAT1 antibody [EPR23049-111] - ChIP Grade

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-STAT1 antibody [EPR23049-111] - ChIP Grade (AB239360)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized HeLa (human cervix adenocarcinoma epithelial cell) cells labelling STAT1 with ab239360 at 1/50 dilution, followed by ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary antibody at 1\1000 dilution (Green). Confocal image showing the signal translocate to nuclear in HeLa cells treated with IFN-γ (10ng/ml) for 30 mins. ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control : Secondary antibody is ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary at 1/1000 dilution.

• ChIP

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ChIP - Anti-STAT1 antibody [EPR23049-111] - ChIP Grade (AB239360)

Chromatin was prepared from HT-1080 cells treated with IFN-γ (50ng/ml 30min) according to the Abcam Dual-X-ChIP protocol*. Cells were fixed with 1.5 mM EGS for 30mins and then formaldehyde for 10min.

The ChIP was performed with 25 μg of chromatin, 5 μg of ab239360 (red), or 5 μg of rabbit normal IgG ab172730 (gray) and 20 μl of Protein A/G sepharose beads. 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 commercial primers from Cell Signaling Technology (Cat. No. : #5148)

*http : //www.abcam.com/resources?keywords=X%20ChIP%20protocol

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-STAT1 antibody [EPR23049-111] - ChIP Grade (AB239360)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized RAW 264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) cells labelling STAT1 with ab239360 at 1/50 dilution, followed byab150077 AlexaFluor^®488 Goat anti-Rabbit secondary antibody at 1/1000 dilution (Green). Confocal image showing the signal translocate to nuclear in RAW 264.7 cells treated with IFN-γ (10ng/ml) for 30 mins. ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control : Secondary antibody is ab150077 AlexaFluor®488 Goat anti-Rabbit secondary at 1/1000 dilution.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-STAT1 antibody [EPR23049-111] - ChIP Grade (AB239360)

Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol permeabilized RAW264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) cells labelling STAT1 with ab239360 at 1/600 (Red) compared with a Rabbit monoclonal IgG (ab172730)isotype control (black)and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 was used as the secondary antibody.

• IP

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Immunoprecipitation - Anti-STAT1 antibody [EPR23049-111] - ChIP Grade (AB239360)

STAT1 was immunoprecipitated from 0.35mg RAW264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysate with ab239360 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab239360 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1/5000 dilution.

Lane 1 : RAW264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysate (10ug)

Lane 2 : ab239360 IP in RAW264.7 whole cell lysate

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab239360 in RAW264.7 whole cell lysate

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

Exposure time : 75 seconds.

All lanes:

Immunoprecipitation - Anti-STAT1 antibody [EPR23049-111] - ChIP Grade (ab239360)

Predicted band size: 87 kDa

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• WB

Lab

Western blot - Anti-STAT1 antibody [EPR23049-111] - ChIP Grade (AB239360)

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

Lanes 5-7 were developed using a higher sensitivity ECL substrate.

Exposure time : Lanes 1-4 : 3 minutes; Lane 5-7 : 48 seconds.

All lanes:

Western blot - Anti-STAT1 antibody [EPR23049-111] - ChIP Grade (ab239360) at 1/1000 dilution

Lane 1:

Human lymph node tissue lysate at 20 µg

Lane 2:

RAW264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage), whole cell lysate at 20 µg

Lane 3:

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

Lane 4:

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

Lane 5:

Mouse lung tissue lysate at 20 µg

Lane 6:

Mouse breast tissue lysate at 20 µg

Lane 7:

NIH/3T3 (mouse embryonic fibroblast), 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

Predicted band size: 87 kDa

Observed band size: 88 kDa

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