Anti-STAT1 antibody [EPR4407]

7 product images

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  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 (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 Human STAT1 knockout HeLa cell line ab255346 (knockout cell lysate Human STAT1 knockout HeLa 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 (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 (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye^®680RD) preadsorbed (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 (Human STAT1 knockout HeLa cell line ab255346)

  Performed under reducing conditions.

  Predicted band size: 48 kDa, 87 kDa

  Observed band size: 50 kDa, 87 kDa

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  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) (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).

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  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 109320 at 1/50 dilution (2μg). VeriBlot for IP Detection Reagent (HRP)(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 (Rabbit IgG, monoclonal [EPR25A] - Isotype Control 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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  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) (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) 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.

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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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  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.

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  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 Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730 is also shown.
  
  Additional screenshots of mapped reads can be found in the Protocol booklet in the Support and downloads section.
  
  The University of Geneva owns patents relevant to ChIC (Chromatin Immuno-Cleavage) methods.