Lane 1: Wild-type HAP1 cell lysate (20 µg)
Lane 2: KDM1 / LSD1 knockout HAP1 cell lysate (20 µg)
Lane 3: HeLa cell lysate (20 µg)
Lane 4: Jurkat cell lysate (20 µg)
Lanes 1 - 4: Merged signal (red and green). Green - ab90966 observed at 110 kDa. Red - loading control, ab181602, observed at 37 kDa.

ab90966 was shown to specifically recognize KDM1/LSD1 in wild-type HAP1 cells along with additional cross reactive bands. No band was observed when KDM1/LSD1 knockout samples were used. Wild-type and KDM1 / LSD1 knockout samples were subjected to SDS-PAGE. ab90966 and ab181602 (loading control to GAPDH) were diluted to 1/500 and 1/10,000 respectively and incubated overnight at 4°C. Blots were developed with Goat anti-Mouse IgG H&L (IRDye^® 800CW) preadsorbed (ab216772) and Goat Anti-Rabbit IgG H&L (IRDye^® 680RD) preadsorbed (ab216777) secondary antibodies at 1/10,000 dilution for 1hr at room temperature before imaging.

ab90966 staining KDM1A in wild-type HAP1 cells (top panel) and KDM1A knockout HAP1 cells (bottom panel). 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 with ab90966 at 1μg/ml concentration and ab202272 at 1/250 dilution (shown in pseudo colour red) overnight at +4°C, followed by a further incubation at room temperature for 1h with a goat secondary antibody to Mouse IgG (Alexa Fluor® 488) (ab150117) at 2 μg/ml (shown in green). Nuclear DNA was labelled in blue with DAPI.

Ab90966 was tested for immunostaining (upper panel) and IP-Western (lower panel). For immunostaining analysis, HeLa cells were transfected with a shLSD1 plasmid also encoding GFP for two days followed by immunostaining using ab90966. For IP-Western analysis, HeLa nuclear extracts (~200ug for each reaction) were immunoprecipitated with or without 0.5ug LSD antibody followed by Western analysis at a dilution of 1:50.

Overlay histogram showing HeLa cells stained with ab90966 (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 (ab90966, 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 HeLa cells fixed with 4% paraformaldehyde (10 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.