Recombinant Anti-STAT3 antibody [EPR361] (ab109085)

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

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.

Blocking/Diluting buffer: 5% NFDM/TBST.

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

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.

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.

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.

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.

Blocking buffer: 5% NFDM/TBST

Dilution buffer: 5% NFDM/TBST