重组Anti-STAT1抗体[EPR4407]

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-STAT1 antibody [EPR4407] (AB109320)

Overlay histogram showing HeLa cells stained with ab109320 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab109320, 1/10000 dilution) for 30 min at 22°C. The secondary antibody used was Alexa Fluor^® 488 goat anti-rabbit IgG (H&L) (ab150077) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (0.1μg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-STAT1 antibody [EPR4407] (AB109320)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized MCF-7(Human breast adenocarcinoma cell line) cell line labeling STAT1 with ab109320 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green).

Confocal image showing nuclear and cytoplasmic staining on MCF7 cells

The nuclear counterstain is DAPI (blue).

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-STAT1 antibody [EPR4407] (AB109320)

ab109320 at 1/100 dilution staining STAT1 in Human ovary carcinoma by Immunohistochemistry, Paraffin-embedded tissue.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

• IP

Lab

Immunoprecipitation - Anti-STAT1 antibody [EPR4407] (AB109320)

STAT1 was immunoprecipitated from 0.35 mg MCF7 (Human breast adenocarcinoma epithelial cell) whole cell lysate 10 µg with ab109320 at 1/50 dilution (2µg). VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1/5000 dilution.

Lane 1 : MCF7 (Human breast adenocarcinoma epithelial cell) whole cell lysate 10 µg
Lane 2 : ab109320 IP in MCF7 whole cell lysate
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab109320 in MCF7 whole cell lysate

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

All lanes:

Immunoprecipitation - Anti-STAT1 antibody [EPR4407] (ab109320)

Predicted band size: 87 kDa

Observed band size: 90 kDa

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• WB

Lab

Western blot - Anti-STAT1 antibody [EPR4407] (AB109320)

Lanes 1- 2 : Merged signal (red and green). Green - ab109320 observed at 87 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

ab109320 was shown to react with STAT1 in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab255346 (knockout cell lysate ab263837) was used. Wild-type HeLa and STAT1 knockout HeLa cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab109320 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 10000 dilution and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^®800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^®680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-STAT1 antibody [EPR4407] (ab109320) at 1/10000 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: 48 kDa,87 kDa

Observed band size: 50 kDa,87 kDa

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• ChIC/CUT&RUN-seq

Supplier Data

ChIC/CUT&RUN sequencing - Anti-STAT1 antibody [EPR4407] (AB109320)

CUT&RUN profiling with STAT1 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 antibody (Abcam ab109320, 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 ChAsE (Younesy et al., Bioinformatics 2016; PMID 27378294). Row-linked data are ranked by intensity relative to STAT1, with red indicating high localized enrichment and blue denoting background.

• ChIC/CUT&RUN-seq

Supplier Data

ChIC/CUT&RUN sequencing - Anti-STAT1 antibody [EPR4407] (AB109320)

CUT&RUN profiling with STAT1 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 antibody (Abcam ab109320, 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).

• ChIC/CUT&RUN-seq

Supplier Data

ChIC/CUT&RUN sequencing - Anti-STAT1 antibody [EPR4407] (AB109320)

ChIC/CUT&RUN was performed using a pAG-MNAse at a final concentration of 700 ng/mL, 2 x 10^5 HeLa (Human cervix adenocarcinoma epithelial cell line) cells treated with IFN gamma (50ng/ml 1h) and 5µg of ab109320 [EPR4407]. The resulting DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 10 million reads. The negative IgG control ab172730 is also shown. Additional screenshots of mapped reads can be found in the Protocol booklet in the Product Protocol section. The University of Geneva owns patents relevant to ChIC (Chromatin Immuno-Cleavage) methods.

• WB

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Western blot - Anti-STAT1 antibody [EPR4407] (AB109320)

All lanes:

Western blot - Anti-STAT1 antibody [EPR4407] (ab109320) at 1/10000 dilution

Lane 1:

293T cell lysate at 10 µg

Lane 2:

HeLa cell lysate at 10 µg

Lane 3:

MCF7 cell lysate at 10 µg

Predicted band size: 87 kDa

Observed band size: 90 kDa

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