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.