All lanes : Anti-STAT3 antibody [EPR361] (ab109085) at 1/1000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : STAT3 knockout HeLa cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 88 kDa
Observed band size: 92 kDa why is the actual band size different from the predicted?

Lanes 1- 2: Merged signal (red and green). Green - ab109085 observed at 92 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

 ab109085 was shown to react with STAT3 in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab255436 (knockout cell lysate ab263797) was used. Wild-type HeLa and STAT3 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. ab109085 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 1000 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.

Immunohistochemical staining of paraffin embedded human pancreas with purified ab109085 at a working dilution of 1 in 100. The secondary antibody used is a HRP polymer for rabbit IgG. The sample is counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0. PBS was used instead of the primary antibody as the negative control, and is shown in the inset.

Immunocytochemistry/Immunofluorescence analysis of HeLa (Human epithelial cell line from cervix adenocarcinoma) labeling STAT3 with Purified ab109085 at 1/250 dilution (5 µg/ml). Cells were fixed with 4% PFA and permeabilized with 0.1% tritonX-100. ab150077 Goat anti rabbit IgG(Alexa Fluor^® 488) at 1/1000 dilution was used as the secondary antibody. Nuclei were counterstained with DAPI. PBS was used instead of the primary antibody as the negative control.

All lanes : Anti-STAT3 antibody [EPR361] (ab109085) at 1/1000 dilution

Lane 1 : HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate
Lane 2 : HaCaT (Human skin keratinocyte) whole cell lysate
Lane 3 : NIH/3T3 (Mouse embryonic fibroblast) whole cell lysate
Lane 4 : C2C12 (Mouse myoblasts cell line) whole cell lysate
Lane 5 : Human brain lysate
Lane 6 : Mouse brain lysate
Lane 7 : Rat brain lysate
Lane 8 : Rat liver lysate

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: 88 kDa
Observed band size: 92 kDa why is the actual band size different from the predicted?
Additional bands at: 32 kDa, 40 kDa. We are unsure as to the identity of these extra bands.


Exposure time: 50 seconds

Blocking/Diluting buffer: 5% NFDM/TBST.

Rabbit monoclonal [EPR16891] to GAPDH (ab181602) used as loading control.

All lanes : Anti-STAT3 antibody [EPR361] (ab109085) at 1/1000 dilution

Lane 1 : Wild-type HAP1 whole cell lysate
Lane 2 : STAT3 knockout HAP1 whole cell lysate
Lane 3 : HeLa whole cell lysate
Lane 4 : HEK293 whole cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 88 kDa

Lanes 1 - 4: Merged signal (red and green). Green - ab109085 observed at 92 kDa. Red - loading control, ab8245, observed at 37 kDa.

Ab109085 was shown to specifically react with STAT3 in wild-type cells as signal was lost in STAT3 knockout HAP1 cells. Wild-type and STAT3 knockout samples were subjected to SDS-PAGE. Ab109085 and ab8245 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at 1/1000 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.

Flow cytometry analysis of HeLa (human cervix adenocarcinoma) cells labeling STAT3 (red) with ab109085 at a 1/300 dilution. Cells were fixed with 4% paraformaldehyde and permeabilized with 90% methanol. A goat anti-rabbit IgG (Alexa Fluor^® 488) (ab150077) was used as the secondary antibody at a 1/2000 dilution. Black - Rabbit monoclonal IgG (ab172730). Blue (unlabeled control) - Cells without incubation with the primary and secondary antibodies.

All lanes : Anti-STAT3 antibody [EPR361] (ab109085) at 1/2000 dilution (purified)

Lane 1 : Mouse heart tissue lysate
Lane 2 : Rat brain tissue lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : HRP goat anti-rabbit (H+L) at 1/1000 dilution

Predicted band size: 88 kDa
Observed band size: 88 kDa

Blocking buffer: 5% NFDM/TBST

Dilution buffer: 5% NFDM/TBST

Immunofluorescence staining of A549 cells with purified ab109085 at a working dilution of 1 in 150, counter-stained with DAPI. The secondary antibody was Alexa Fluor^® 555 goat anti rabbit (ab150078), used at a dilution of 1 in 500. The cells were fixed in 4% PFA and permeabilized using 0.1% Triton X 100. The negative control is shown in bottom right hand panel - for the negative control, purified ab109085 was used at a dilution of 1/200 followed by an Alexa Fluor^® 488 goat anti-mouse antibody at a dilution of 1/500.

All lanes : Anti-STAT3 antibody [EPR361] (ab109085) at 1/2000 dilution (purified)

Lane 1 : A431 cell lysate
Lane 2 : Raji cell lysate
Lane 3 : HeLa cell lysate
Lane 4 : SH-S5SY (Human neuroblastoma cell line from bone marrow) cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : HRP goat anti-rabbit (H+L) at 1000 mg/ml

Predicted band size: 88 kDa
Observed band size: 88 kDa

Blocking buffer: 5% NFDM/TBST

Dilution buffer: 5% NFDM/TBST

All lanes : Anti-STAT3 antibody [EPR361] (ab109085) at 1/1000 dilution (unpurified)

Lane 1 : A431 cell lysate
Lane 2 : Raji cell lysate
Lane 3 : HeLa cell lysate
Lane 4 : SH-SY5Y cell lysate

Lysates/proteins at 10 µg per lane.

Predicted band size: 88 kDa