Lane 1: Wild type HAP1 whole cell lysate (20 µg)
Lane 2: Empty (0 µg)
Lane 3: BCL11A (Ctip1) knockout HAP1 whole cell lysate (20 µg)
Lane 4: Raji whole cell lysate (20 µg)

Lanes 1 - 4: Merged signal (red and green). Green - ab19489 observed at 91 kDa. Red - loading control, ab181602, observed at 37 kDa.

ab19489 was shown to recognize BCL11A (Ctip1) in wild type cells as signal was lost at the expected MW in BCL11A (Ctip1) knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and Empty knockout samples were subjected to SDS-PAGE. ab19489 and ab181602 (Rabbit anti GAPDH loading control) were incubated overnight at 4°C at 1000 dilution and 1/10000 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 1/10000 dilution for 1 hour at room temperature before imaging.

Human normal tonsil. Staining is localised to cytoplasm and nuclei. Left panel: with primary antibody at 4 ug/ml. Right panel: isotype control. Sections were stained using an automated system DAKO Autostainer Plus , at room temperature: sections were rehydrated and antigen retrieved with the Dako 3 in 1 AR buffers citrate EDTA pH 9.0 in a DAKO PT Link. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 mins. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 min and detected with Dako envision flex amplification kit for mouse for 30 minutes. Colorimetric detection was completed with Diaminobenzidine for 5 minutes. Slides were counterstained with Haematoxylin and coverslipped under DePeX. Please note that for manual staining we recommend to optimize the primary antibody concentration and incubation time (overnight incubation), and amplification may be required.

Overlay histogram showing Ramos cells stained with ab19489 (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 (ab19489, 2µg/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 Ramos cells fixed with 4% paraformaldehyde (10 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.