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

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.

Immunohistochemical analysis of paraffin-embedded lung squamous carcinoma using ab32500 at a 1/50 dilution.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

ab32500 staining STAT3 in HeLa cells. The cells were fixed with 4% formaldehyde (10min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated overnight at +4°C with ab32500 at 1/250 dilution (shown in green) and ab195889, Mouse monoclonal [DM1A] to alpha Tubulin (Alexa Fluor® 594), at 2µg/ml (shown in red). Nuclear DNA was labelled with DAPI (shown in blue).
Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

Overlay histogram showing HeLa cells stained with ab32500 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween 20 for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab32500, 1/100) for 30 min at 22°C. The secondary antibody used was Alexa Fluor^® 488 goat anti-rabbit IgG (H&L) (ab150081) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (ab172730, 1µg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. 

Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter. 

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.