重组Anti-STAT3抗体[EPR787Y] (ab68153)

Lanes 1- 2: Merged signal (red and green). Green - ab68153 observed at 92 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

 ab68153 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. ab68153 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.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human brain tissue sections labelling STAT3 with purified ab68153 at 1/200. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. A prediluted HRP-polymer conjugated anti-rabbit IgG was used as the secondary antibody. Counterstained with Hematoxylin.

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 - ab68153 observed at 92 kDa. Red - loading control, ab8245, observed at 37 kDa.

Ab68153 detected the expected band for STAT3 in wild-type cells along with additional cross-reactive bands. The band was not seen in STAT3 knockout HAP1 cells. Wild-type and STAT3 knockout samples were subjected to SDS-PAGE. Ab68153 and ab8245 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at 1/500 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.

Immunocytochemistry/Immunofluorescence analysis of HeLa cells labelling STAT3 (green) with purified ab68153 at 1/200. Cells were fixed with 4% paraformaldehyde. An Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/200) was used as the secondary antibody. DAPI (blue) was used as the nuclear counterstain.

ab68153 staining STAT3 in the human cell line HeLa (human cervix adenocarcinoma) by flow cytometry. Cells were fixed with 4% paraformaldehyde, permeabilized with 90% methanol and the sample was incubated with the primary antibody at a dilution of 1/30. A goat anti rabbit IgG (Alexa Fluor® 488) at a dilution of 1/2000 was used as the secondary antibody.

Isoytype control: Rabbit monoclonal IgG (Black)

Unlabelled control: Cell without incubation with primary antibody and secondary antibody (Blue)

Blocking buffer and concentration: 5% NFDM/TBST.

Diluting buffer and concentration: 5% NFDM /TBST.

Flow cytometry analysis of Raji cells labelling STAT3 with unpurified ab68153 at 1/30 (red). Cells were fixed with 2% paraformaldehyde. A FITC-conjugated goat anti-rabbit IgG (1/150) was used as the secondary antibody. A rabbit monoclonal IgG was used as the isotype control (green).

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human brain tissue sections labelling STAT3 with unpurified ab68153 at 1/140. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. A prediluted HRP-polymer conjugated anti-rabbit IgG was used as the secondary antibody. Counterstained with Hematoxylin.

Immunocytochemistry/Immunofluorescence analysis of HeLa cells labelling STAT3 (green) with unpurified ab68153 at 1/140. Cells were fixed with 4% paraformaldehyde. An Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/200) was used as the secondary antibody. DAPI (blue) was used as the nuclear counterstain.