All lanes : Anti-Notch1 antibody [mN1A] (ab128076) at 1/1000 dilution

Lane 1 : Wild-type HAP1 whole cell lysate
Lane 2 : NOTCH1 knockout HAP1 whole cell lysate

Lysates/proteins at 40 µg per lane.

Predicted band size: 272 kDa

Lanes 1 - 2: Merged signal (red and green). Green - ab128076 observed at 110 kDa. Red - loading control, ab181602, observed at 37 kDa.

ab128076 was shown to recognize NOTCH1 in wild-type HAP1 cells as signal was lost at the expected MW in NOTCH1 knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and NOTCH1 knockout samples were subjected to SDS-PAGE. The membrane was blocked with 3% Milk. Ab128076 and ab181602 (Rabbit anti-GAPDH loading control) were incubated overnight at 4°C at 1/1000 dilution 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.

Overlay histogram showing SH-SY5Y cells stained with ab128076 (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 (ab128076, 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. This antibody gave a positive signal in SH-SY5Y cells fixed with 4% paraformaldehyde (10 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.