All lanes : Anti-STAT1 antibody [EPR4407] (ab109320) at 1/10000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : STAT1 knockout HeLa cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 87 kDa
Observed band size: 87 kDa

Lanes 1- 2: Merged signal (red and green). Green - ab109320 observed at 87 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

 ab109320 was shown to react with STAT1 in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab255346 (knockout cell lysate ab263837) was used. Wild-type HeLa and STAT1 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. ab109320 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 10000 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.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized MCF-7(Human breast adenocarcinoma cell line) cell line  labeling STAT1 with ab109320 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green).

Confocal image showing nuclear and cytoplasmic staining on MCF7 cells

The nuclear counterstain is DAPI (blue).

Overlay histogram showing HeLa cells stained with ab109320 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween 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 (ab109320, 1/10000 dilution) for 30 min at 22°C. The secondary antibody used was Alexa Fluor® 488 goat anti-rabbit IgG (H&L) (ab150077) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (0.1μg/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.

All lanes : Anti-STAT1 antibody [EPR4407] (ab109320) at 1/10000 dilution

Lane 1 : 293T cell lysate
Lane 2 : HeLa cell lysate
Lane 3 : MCF7 cell lysate

Lysates/proteins at 10 µg per lane.

Predicted band size: 87 kDa
Observed band size: 90 kDa why is the actual band size different from the predicted?

ab109320 at 1/100 dilution staining STAT1 in Human ovary carcinoma by Immunohistochemistry, Paraffin-embedded tissue.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.