All lanes : Anti-STAT1 antibody [EPR21057-141] - ChIP Grade (ab234400) at 1/1000 dilution

Lane 1 : Wild-type HAP1 whole cell lysate
Lane 2 : STA1 knockout HAP1 whole cell lysate
Lane 3 : HeLa whole cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 87 kDa

Lanes 1 - 3: Merged signal (red and green). Green - ab234400 observed at 87 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab234400 was shown to specifically react with STAT1 in wild-type HAP1 cells as signal was lost in STA1 knockout cells. Wild-type and STA1 knockout samples were subjected to SDS-PAGE. The membrane was blocked with 3% Milk. Ab234400 and ab8245 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1/1000 dilution and 1/20000 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 1/20000 dilution for 1 hour at room temperature before imaging.

All lanes : Anti-STAT1 antibody [EPR21057-141] - ChIP Grade (ab234400) at 1/1000 dilution

Lane 1 : HeLa (human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 20 µg
Lane 2 : HEK-293T (human epithelial cell line from embryonic kidney transformed with large T antigen) whole cell lysate at 20 µg
Lane 3 : MCF7 (human breast adenocarcinoma cell line) whole cell lysate at 20 µg
Lane 4 : Human heart tissue lysate at 10 µg

Secondary
Lanes 1-3 : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution
Lane 4 : VeriBlot for IP Detection Reagent (HRP) (ab131366) at 1/4000 dilution

Predicted band size: 87 kDa

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

Exposure time: Lanes 1-2 & 4: 3 minutes; Lane 3: 15 seconds.

The doublet observed in some lanes likely represent the α and β isoforms of Stat1 (PMID: 8647800).

Immunohistochemical analysis of paraffin-embedded human tonsil tissue labeling STAT1 with ab234400 at 1/5000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) ready to use. Positive staining mainly on interfollicular cells of human tonsil (PMID: 25336386; PMID: 25921060) is observed. Counter stained with hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) ready to use.

Heat mediated antigen retrieval was performed with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol permeabilized HeLa (human epithelial cell line from cervix adenocarcinoma) cells labeling STAT1 with ab234400 at 1/500 (Red) compared with Rabbit monoclonal IgG (ab172730) (Black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

STAT1 was immunoprecipitated from 0.35 mg of MCF7 (human breast adenocarcinoma cell line) whole cell lysate with ab234400 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab234400 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used for detection antibody at 1/5000 dilution.
Lane 1: MCF7 whole cell lysate 10 μg (input).
Lane 2: ab234400 IP in MCF7 whole cell lysate.
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab234400 in MCF7 whole cell lysate.

Blocking and dilution buffer and concentration: 5% NFDM/TBST.
Exposure time: 3 minutes.

The doublet observed in some lanes likely represent the α and β isoforms of STAT1 (PMID: 8647800).

Chromatin was prepared from HeLa (starve overnight) + hIFN-α (serum-starved overnight) (1,000 units/ml,30 min) 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 ab234400 (red), and 20 µl of protein A/G sepharose beads slurry (10 µl of sepharose A beads + 10 µl of sepharose G beads). 2 μg of rabbit normal IgG was added to the beads control (gray). The immunoprecipitated DNA was quantified by real time PCR (SYBR approach).
ChIP results are consistent with the literature (PMID: 16319195).

Immunohistochemical analysis of paraffin-embedded human kidney tissue labeling STAT1 with ab234400 at 1/5000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) ready to use. Positive staining on glomerulus and some renal tubules of human kidney (PMID: 26678048) is observed. Counter stained with hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) ready to use.

Heat mediated antigen retrieval was performed with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

 

Flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol permeabilized MCF7 (human breast adenocarcinoma cell line) cells labeling STAT1 with ab234400 at 1/500 (Red) compared with Rabbit monoclonal IgG (ab172730) (Black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

Immunohistochemical analysis of paraffin-embedded paired human endometrial cancer (A) and non-tumor endometrium tissue (B) labeling STAT1 with ab234400 at 1/5000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) ready to use. Much higher staining intensity of endometrial cancer (A) than its paired non-tumor endometrium (B) (PMID: 25267067) is observed. Counter stained with hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) ready to use.

Heat mediated antigen retrieval was performed with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.