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).

Chromatin was prepared from HepG2 + IL-6 (100 ng/ml 30 minutes) cells according to the Abcam X-ChIP protocol*. Cells were fixed with formaldehyde for 10 minutes.
The ChIP was performed with 25 µg of chromatin, 2 µg of ab32500 (red), and 20 µl of Protein A/G sepharose beads. 2 µg of rabbit normal IgG was added to the beads control (grey). The immunoprecipitated DNA was quantified by real time PCR (Sybr green approach).
Primers and probes are located in the first kb of the transcribed region.
*http///www.abcam.com/resources?keywords=X%20ChIP%20protocol

ab32500 (purified) at 1/60 dilution (2.594 µg/ml) 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 1/500 and VeriBlot for IP secondary antibody (HRP) (ab131366) was used at 1/1000 dilution.

Blocking and diluting buffer: 5% NFDM /TBST.

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 22ºC. The secondary antibody used was Alexa Fluor® 488 goat anti-rabbit IgG (H&L) (ab150081) at 1/2000 dilution for 30 min at 22ºC. 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.

Anti-STAT3 antibody [E121-21] - ChIP Grade (ab32500) at 1/1000 dilution + A431 cell lysate

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