Recombinant Anti-STAT3 antibody [E121-21] (ab32500)

Flow Cytometry analysis of A431 (Human epidermoid carcinoma epithelial cell) cells labelling STAT3 with Purified ab32500 at 1:20 dilution (5 µg/ml) (Red). Cells were fixed with 4% Paraformaldehyde and permeabilised with 90% Methanol. A Goat anti rabbit IgG (Alexa Fluor® 488, ab150081) secondary antibody was used at 1:2000. Isotype control - Rabbit monoclonal IgG (Black). Unlabelled control - Cell without incubation with primary antibody and secondary antibody (Blue).

Immunocytochemistry analysis of SH-SY5Y (Human neuroblastoma epithelial cell) cells labeling STAT3 with Purified ab32500 at 1:50 dilution (2.1 µg/ml). Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. 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. PBS instead of the primary antibody was used as the secondary antibody only control.

Lane 1: Wild type HAP1 whole cell lysate (20 µg)
Lane 2: STAT3 knockout HAP1 whole cell lysate (20 µg)
Lane 3: HeLa whole cell lysate (20 µg)
Lane 4: HEK293 whole cell lysate (20 µg)

Lanes 1 - 4: Merged signal (red and green). Green - ab32500 observed at 92 kDa. Red - loading control, ab8245, observed at 37 kDa.

Ab32500 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. Ab32500 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.

ab32500 at 1/20 dilution (0.5 µg) immunoprecipitating STAT3 in HeLa whole cell lysate.

Lane 1 (input): HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate 10µg
Lane 2 (+): ab32500 & HeLa whole cell lysate
Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab32500 in HeLa whole cell lysate

For western blotting, ab32500 at 1/1000 and VeriBlot for IP secondary antibody (HRP) (ab131366) was used at 1/5000 dilution.

Blocking and diluting buffer: 5% NFDM /TBST.