Lane 1: Wild-type HAP1 whole cell lysate (20 µg)
Lane 2: RUNX3 knockout HAP1 whole cell lysate (20 µg)
Lane 3: HeLa whole cell lysate (20 µg)

Lanes 1 - 3: Merged signal (red and green). Green - ab40278 observed at 44 kDa. Red - loading control, ab176560, observed at 50 kDa.

ab40278 was shown to specifically react with RUNX3 in wild-type HAP1 cells as signal was lost in RUNX3 knockout cells. Wild-type and RUNX3 knockout samples were subjected to SDS-PAGE. ab40278 and ab176560 (Rabbit anti-alpha Tubulin loading control) were incubated overnight at 4°C at 1 μg/ml and 1/20000 dilution respectively. Blots were developed with Goat anti-Mouse IgG H&L (IRDye^® 800CW) preabsorbed ab216772 and Goat anti-Rabbit IgG H&L (IRDye^® 680RD) preabsorbed ab216777 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

IHC image of RUNX3 staining in human lymphoma FFPE section, performed on a Bond^TM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab40278, 1µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

Overlay histogram showing HEK293 cells stained with ab40278 (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 (ab40278, 1µg/1x10⁶ cells) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed.

All lanes : Anti-RUNX3 antibody [R3-5G4] (ab40278) at 1 µg/ml

Lane 1 : Jurkat (Human T cell lymphoblast-like cell line) Whole Cell Lysate
Lane 2 : Raji (Human Burkitt's lymphoma cell line) Whole Cell Lysate
Lane 3 : SW480 whole cell lysate (ab3957)

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : IRDye 680 Conjugated Rabbit Anti-Mouse IgG (H+L) at 1/10000 dilution

Performed under reducing conditions.

Predicted band size: 44 kDa
Additional bands at: 46 kDa (possible isoform)