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 - ab19487 observed at 91 kDa. Red - loading control, ab181602, observed at 37 kDa.

ab19487 was shown to specifically react with BCL11A (Ctip1) in HAP1 wild type cells. No band was observed when BCL11A (Ctip1) knockout cells were examined. Wild-type and BCL11A (Ctip1) knockout samples were subjected to SDS-PAGE. ab19487 and ab181602 (Rabbit anti GAPDH loading control) were incubated overnight at 4°C at 1/1000 dilution and 1/10,000 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 1/10,000 dilution for 1 hour at room temperature before imaging.

All lanes : Anti-Ctip1/BCL-11A antibody [14B5] (ab19487)

Lane 1 : C6 Cell Line
Lane 2 : MEF Cell Line
Lane 3 : HDFn Cell LIne
Lane 4 : Jurkat Cell Line
Lane 5 : HEK293T Cell Line

Lysates/proteins at 20 µg per lane.

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 91 kDa

The CTIP1 / BCL-11A protein band is indicated at 92 kDa. This antibody also identified a 55 kDa protein band in Western blotting, principally in mouse fibroblasts (MEF), but this is unrelated to.

Blocking was performed in 5% milk (in PBS). The primary antibody was diluted in 1% milk (in PBS) and incubated overnight at 4ºC.

Overlay histogram showing Ramos cells stained with ab19487 (red line). The cells were fixed with 4% paraformaldehyde (10 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 (ab19487, 0.1μg/1x10⁶ cells) for 30 min at 22°C. The secondary antibody used was Alexa Fluor® 488 goat anti-mouse IgG (H+L) (ab150113) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 1μg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter. This antibody gave a positive signal in Ramos cells fixed with 80% methanol (5 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

Anti-Ctip1/BCL-11A antibody [14B5] (ab19487) at 1 µg/ml + Raji (Human Burkitt's lymphoma cell line) Whole Cell Lysate at 10 µg

Secondary
Goat Anti-Mouse IgG H&L (HRP) preadsorbed (ab97040) at 1/5000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 91 kDa
Observed band size: 120 kDa why is the actual band size different from the predicted?
Additional bands at: 37 kDa, 42 kDa, 62 kDa. We are unsure as to the identity of these extra bands.


Exposure time: 20 minutes

The 120 kDa band observed is comparable to the molecular weight seen with other commercially available antibodies to Ctip1 / BCL-11A.