重组Anti-STAT1 alpha抗体[EPYR2154]

• IP

Unknown

Immunoprecipitation - Anti-STAT1 alpha antibody [EPYR2154] (AB92506)

ab92506 (purified) at 1/20 dilution (0.5ug) immunoprecipitating STAT1 alpha in A431 whole cell lysate. A431 (Human epidermoid carcinoma epithelial cell) whole cell lysate 10ug
Lane 2 (+) : ab92506 & A431 whole cell lysate
Lane 3 (-) : Rabbit monoclonal IgG (ab172730) instead of ab92506 in A431 whole cell lysate
For western blotting, VeriBlot for IP secondary antibody (HRP) (ab131366) was used at 1/2000 dilution.
Blocking and diluting buffer : 5% NFDM/TBST.

All lanes:

Immunoprecipitation - Anti-STAT1 alpha antibody [EPYR2154] (ab92506)

Predicted band size: 87 kDa

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• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-STAT1 alpha antibody [EPYR2154] (AB92506)

Immunohistochemical analysis of paraffin-embedded Mouse spleen tissue labeling STAT1 alpha with ab92506 at 1/5000 dilution.

Positive staining on mouse spleen

The primary antibody was incubated for 30 mins at room temperature.

The immunostaining was performed on a Leica Biosystems BOND^® RX instrument

Counterstained with Hematoxylin.

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-STAT1 alpha antibody [EPYR2154] (AB92506)

Immunohistochemical analysis of paraffin-embedded (A) Colon tissue from wild-type C57BL/6JGpt mice and (B) Colon tissue from Stat1 konckout mice tissue labeling STAT1 alpha with ab92506 at 1/5000 dilution, followed by a ready to use LeicaDS9800 (Bond^™ Polymer Refine Detection).

Positive staining on (A) Colon tissue from wild-type C57BL/6JGpt mice, no staining on (B) Colon tissue from Stat1 konckout mice.

The primary antibody was incubated for 30 mins at room temperature.

The immunostaining was performed on a Leica Biosystems BOND^® RX instrument.

Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond^™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

The tissue samples were kindly provided by GemPharmatech. C57BL/6JGpt wildtype mice and Stat1-KO homozygous mice (Strain ID : T006005).

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-STAT1 alpha antibody [EPYR2154] (AB92506)

Immunohistochemical analysis of paraffin-embedded (A) Cerebrum tissue from wild-type C57BL/6JGpt mice and (B) Cerebrum tissue from Stat1 konckout mice tissue labeling STAT1 alpha with ab92506 at 1/5000 dilution, followed by a ready to use LeicaDS9800 (Bond^™ Polymer Refine Detection).

Positive staining on (A) Cerebrum tissue from wild-type C57BL/6JGpt mice, no staining on (B) Cerebrum tissue from Stat1 konckout mice.

The primary antibody was incubated for 30 mins at room temperature.

The immunostaining was performed on a Leica Biosystems BOND^® RX instrument.

Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond^™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

The tissue samples were kindly provided by GemPharmatech. C57BL/6JGpt wildtype mice and Stat1-KO homozygous mice (Strain ID : T006005).

• WB

Lab

Western blot - Anti-STAT1 alpha antibody [EPYR2154] (AB92506)

Phospho STAT1 protein is revealed by ab109461, anti-STAT1 (phospho S727) antibody. ab92506 is used as pan control which detects total STAT1.

Blocking and dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-STAT1 alpha antibody [EPYR2154] (ab92506) at 1/2000 dilution

Lane 1:

Untreated HeLa (human cervix adenocarcinoma) membrane at 10 µg

Lane 2:

HeLa (human cervix adenocarcinoma) membrane treated with Alkaline Phosphatase at 10 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1500 dilution

Predicted band size: 87 kDa

Observed band size: 91 kDa

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Exposure time: 3min

• WB

Lab

Western blot - Anti-STAT1 alpha antibody [EPYR2154] (AB92506)

Lane 1 : Wild type HAP1 whole cell lysate (20 μg)
Lane 2 : STAT1 alpha knockout HAP1 whole cell lysate (20 μg)
Lane 3 : HeLa whole cell lysate (20 μg)
Lane 4 : A431 whole cell lysate (20 μg)

Lanes 1 - 4 : Merged signal (red and green). Green - ab92506 (unpurified) observed at 90 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab92506 was shown to specifically react with STAT1 alpha when STAT1 alpha knockout samples were used. Wild-type and STAT1 alpha knockout samples were subjected to SDS-PAGE. ab92506 and ab8245 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at 1/500 dilution and 1/10000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-STAT1 alpha antibody [EPYR2154] (ab92506)

Predicted band size: 87 kDa

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• WB

Lab

Western blot - Anti-STAT1 alpha antibody [EPYR2154] (AB92506)

Lanes 1 - 2 : Merged signal (red and green). Green - ab92506 observed at 85 kDa. Red - loading control ab7291 (Mouse anti-Alpha Tubulin [DM1A]) observed at 55 kDa.

ab92506 was shown to react with STAT1 alpha in wild-type HeLa cells in Western blot with loss of signal observed in STAT1 knockout cell line ab255346 (STAT1 knockout cell lysate ab263837). Wild-type HeLa and STAT1 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3 % milk in TBS-T (0.1 % Tween^®) before incubation with ab92506 and ab7291 (Mouse anti-Alpha Tubulin [DM1A]) overnight at 4 °C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 h at room temperature before imaging.

All lanes:

Western blot - Anti-STAT1 alpha antibody [EPYR2154] (ab92506) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

STAT1 knockout HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human STAT1 knockout HeLa cell line (<a href='/products/cell-lines/human-stat1-knockout-hela-cell-line-ab255346'>ab255346</a>)

Predicted band size: 87 kDa

Observed band size: 85 kDa

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• ChIC/CUT&RUN-seq

Supplier Data

ChIC/CUT&RUN sequencing - Anti-STAT1 alpha antibody [EPYR2154] (AB92506)

CUT&RUN profiling with STAT1 alpha antibody demonstrates robust genome-wide enrichment in cells. Heatmaps of genome-wide signal flanking annotated transcription start sites (TSSs, +/- 2 kbp) display CUT&RUN data generated using the CUTANA™ CUT&RUN Kit (EpiCypher 14-1048) with STAT1 alpha antibody (Abcam ab92506, 0.5 µg). 500,000 HeLa cells were used per reaction. IgG antibody was included as a negative control to assess non-specific background. Libraries were prepared using the CUTANA™ CUT&RUN Library Prep Kit (EpiCypher 14-1001). Sequencing was performed with paired-end 50 bp reads, and data were processed on CUTANA™ Cloud (cloud.epicypher.com) by alignment to the hg38 genome. Heatmaps were generated using deepTools (Ramнrez et al., Nucleic Acids Res. 2014; PMID : 24799436). Row-linked data are ranked by intensity relative to STAT1 alpha, with red indicating high localized enrichment and blue denoting background.

• ChIC/CUT&RUN-seq

Supplier Data

ChIC/CUT&RUN sequencing - Anti-STAT1 alpha antibody [EPYR2154] (AB92506)

CUT&RUN profiling with STAT1 alpha antibody reveals the expected genomic enrichment pattern in cells. Representative genome browser tracks show CUT&RUN data generated using the CUTANA™ CUT&RUN Kit (EpiCypher 14-1048) with STAT1 alpha antibody (Abcam ab92506, 0.5 µg). 500,000 HeLa cells were used per reaction. IgG, H3K4me3, and H3K27me3 antibodies were included as controls to assess non-specific background, active promoters, and repressed chromatin, respectively. Libraries were prepared using the CUTANA™ CUT&RUN Library Prep Kit (EpiCypher 14-1001). Sequencing was performed with paired-end 50 bp reads, and data were processed on CUTANA™ Cloud (cloud.epicypher.com) by alignment to the hg38 genome. Images were generated using Integrative Genomics Viewer (IGV, Broad Institute).

• WB

Lab

Western blot - Anti-STAT1 alpha antibody [EPYR2154] (AB92506)

Exposure time : Lane 1-4 : 6 second; Lane 5-8 : 15.

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

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

The tissue samples were kindly provided by GemPharmatech. C57BL/6JGpt wildtype mice and Stat1-KO homozygous mice (Strain ID : T006005).

All lanes:

Western blot - Anti-STAT1 alpha antibody [EPYR2154] (ab92506) at 1/1000 dilution

Lane 1:

Wild-type mouse colon tissue lysate (male) at 20 µg

Lane 2:

Wild-type mouse colon tissue lysate (female) at 20 µg

Lane 3:

Stat1 knockout mouse colon tissue lysate (male) at 20 µg

Lane 4:

Stat1 knockout mouse colon tissue lysate (female) at 20 µg

Lane 5:

Wild-type mouse brain tissue lysate (male) at 20 µg

Lane 6:

Wild-type mouse brain tissue lysate (female) at 20 µg

Lane 7:

Stat1 knockout mouse brain tissue lysate (male) at 20 µg

Lane 8:

Stat1 knockout mouse brain tissue lysate (female) at 20 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

Observed band size: 91 kDa

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• WB

Unknown

Western blot - Anti-STAT1 alpha antibody [EPYR2154] (AB92506)

All lanes:

Western blot - Anti-STAT1 alpha antibody [EPYR2154] (ab92506) at 1/1000 dilution

Lane 1:

HeLa cell lysate at 10 µg

Lane 2:

293 cell lysate at 10 µg

Lane 3:

NIH-3T3 cell lysate at 10 µg

Lane 4:

A431 cell lysate at 10 µg

Secondary

All lanes:

HRP labelled goat anti-rabbit IgG at 1/2000 dilution

Predicted band size: 87 kDa

Observed band size: 91 kDa

false

• WB

Lab

Western blot - Anti-STAT1 alpha antibody [EPYR2154] (AB92506)

All lanes:

Western blot - Anti-STAT1 alpha antibody [EPYR2154] (ab92506) at 1/1000 dilution

Lane 1:

A431 (Human epidermoid carcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

Mouse brain lysate at 20 µg

Lane 3:

NIH/3T3 (Mouse embryonic fibroblast) whole cell lysates at 20 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: 87 kDa

Observed band size: 91 kDa

false