All lanes : Anti-STAT2 antibody [Y141] (ab32367) at 1/5000 dilution

Lane 1 : Wild-type A549 cell lysate
Lane 2 : STAT2 knockout A549 cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 98 kDa
Observed band size: 97 kDa why is the actual band size different from the predicted?

Lanes 1 - 2: Merged signal (red and green). Green - ab32367 observed at 97 kDa. Red - loading control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.

ab32367 was shown to react with STAT2 in A549 wild-type cells in western blot with loss of signal observed in STAT2 knockout cell line ab267004 (STAT2 knockout cell lysate ab257183). Wild-type and STAT2 knockout A549 cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% Milk before incubation with ab32367 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4 °C at a 1 in 5000 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.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human hepatocellular carcinoma tissue sections labeling STAT2 with purified ab32367 at 1/100 dilution (6.22 µg/mL). Heat mediated antigen retrieval was performed using Bond™ Epitope Retrieval Solution 2 (pH 9.0) . Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) was used as the secondary antibody. Negative control: PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

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

All lanes : Anti-STAT2 antibody [Y141] (ab32367) at 1/5000 dilution

Lane 1 : Wild-type A549 cell lysate
Lane 2 : STAT2 knockout A549 cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 98 kDa
Observed band size: 97 kDa why is the actual band size different from the predicted?

Lanes 1- 2: Merged signal (red and green). Green - ab32367 observed at 97 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

 ab32367 was shown to react with STAT2 in wild-type A549 cells in western blot. Loss of signal was observed when knockout cell line ab267005 (knockout cell lysate ab257184) was used. Wild-type A549 and STAT2 knockout A549 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. ab32367 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 5000 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 : Anti-STAT2 antibody [Y141] (ab32367) at 1/5000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : STAT2 knockout HeLa cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 98 kDa
Observed band size: 97 kDa why is the actual band size different from the predicted?

Lanes 1- 2: Merged signal (red and green). Green - ab32367 observed at 97 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

 ab32367 was shown to react with STAT2 in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab261819 (knockout cell lysate ab257182) was used. Wild-type HeLa and STAT2 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. ab32367 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 5000 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 : Anti-STAT2 antibody [Y141] (ab32367) at 1/5000 dilution

Lane 1 : Wild-type A549 cell lysate
Lane 2 : STAT2 knockout A549 cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 98 kDa
Observed band size: 97 kDa why is the actual band size different from the predicted?

Lanes 1-2: Merged signal (red and green). Green - ab32367 observed at 97 kDa. Red - loading control ab8245 observed at 37 kDa.

 ab32367 Anti-STAT2 antibody [Y141] was shown to specifically react with STAT2 in wild-type A549 cells. Loss of signal was observed when knockout cell line ab267006 (knockout cell lysate ab257185) was used. Wild-type and STAT2 knockout samples were subjected to SDS-PAGE. ab32367 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 1000 and 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 : Anti-STAT2 antibody [Y141] (ab32367)

Lane 1 : Wild-type HAP1 whole cell lysate
Lane 2 : STAT2 knockout HAP1 whole cell lysate
Lane 3 : K562 whole cell lysate
Lane 4 : THP1 whole cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 98 kDa

Lanes 1 - 4: Merged signal (red and green). Green - ab32367 (unpurified) observed at 97 kDa. Red - loading control, ab9484, observed at 37 kDa.

ab32367 was shown to recognize STAT2 in wild-type HAP1 cells as signal was lost at the expected MW in STAT2 knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and STAT2 knockout samples were subjected to SDS-PAGE. Ab32367 and ab9484 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1/5000 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.

Flow Cytometry analysis of Ramos (Human Burkitt's lymphoma B lymphocyte) cells labeling STAT2 with purified ab32367 at 1/60 dilution (10 µg/mL) (Red). Cells were fixed with 4% Paraformaldehyde and permeabilised with 90% Methanol. A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) secondary antibody was used at 1/2000. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).

Immunocytochemistry/ Immunofluorescence analysis of THP-1 (human monocytic leukemia monocyte) cells labeling STAT2 with purified ab32367 at 1/50 dilution (10 µg/mL). Cells were fixed in 100% Methanol and permeabilized with None. Cells were counterstained with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) 1/200 (2.5 µg/mL). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) was used as the secondary antibody at 1/1000 (2 µg/mL) dilution. DAPI (blue) was used as nuclear counterstain. ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) 1/200 (2.5 µg/mL) was used as the secondary antibody only control.

All lanes : Anti-STAT2 antibody [Y141] (ab32367) at 1/1000 dilution (Purified)

Lane 1 : Ramos (Human Burkitt's lymphoma B lymphocyte) whole cell lysates
Lane 2 : HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysates
Lane 3 : Mouse brain lysates
Lane 4 : Rat brain lysates

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 98 kDa
Observed band size: 113,97 kDa why is the actual band size different from the predicted?

Blocking/Diluting Buffer and concentration: 5% NFDM/TBST

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of rat liver tissue sections labeling STAT2 with purified ab32367 at 1/100 dilution (6.22 µg/mL). Heat mediated antigen retrieval was performed using Bond™ Epitope Retrieval Solution 2 (pH 9.0) . Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) was used as the secondary antibody. Negative control: PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

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

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of mouse liver tissue sections labeling STAT2 with purified ab32367 at 1/100 dilution (6.22 µg/mL). Heat mediated antigen retrieval was performed using Bond™ Epitope Retrieval Solution 2 (pH 9.0) . Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) was used as the secondary antibody. Negative control: PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

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

Immunohistochemical analysis of paraffin-embedded human thyroid cancer using ab32367 (unpurified). Heat mediated antigen retrieval was performed with citrate buffer pH 6 before commencing with IHC staining protocol.