Recombinant Anti-Ctip1/BCL-11A antibody [EPR14943-44] (ab191401)

Lane 1: Wild type HAP1 whole cell lysate (20 µg)
Lane 2: Empty
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 - ab191401 observed at 91 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab191401 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 BCL11A (Ctip1) knockout samples were subjected to SDS-PAGE. Ab191401 and ab8245 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1000 dilution and 1/10000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.

ab191401 (purified) at 1/50 dilution (2.1 µg/mL) immunoprecipitating Ctip1/BCL-11A in Raji whole cell lysate.
Lane 1 (input): Raji(Human Burkitt's lymphoma B lymphocyte) whole cell lysate 10µg
Lane 2 (+): ab191401 & Raji whole cell lysate
Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab191401 in Raji whole cell lysate
For western blotting, ab191401 at 1/500 dilution (2.1 µg/mL) and VeriBlot for IP Detection Reagent (HRP) (ab131366) was used for detection at 1/1000 dilution.
Blocking and diluting buffer: 5% NFDM /TBST .

Immunohistochemical analysis of paraffin-embedded, rat spleen tissue labeling Ctip1/BCL-11A with ab191401 at a 1/100 dilution (19 μg/ml). Counter stained with hematoxylin. In the negative control PBS was used instead of primary antibody.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunofluorescence analysis of, paraformaldehyde-fixed, rat C6 cells labeling Ctip1/BCL-11A with ab191401 at a 1/450 dilution (4 ug/ml). As secondary antibody goat anti-rabbit IgG (Alexa Fluor®488) ab150077 was used at a 1/200 dilution. In blue DAPI staining. In the negative controls cells were treated with anti-BCL11A at a 1/450 dilution as primary antibody and goat anti-mouse IgG (Alexa Fluor®594) at a 1/400 dilution as secondary antibody.

Intracellular Flow Cytometry analysis of Jurkat (human acute T cell leukemia) labelling Ctip1/BCL-11A with purified ab191401 at 1/200 (red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. Alexa Fluor^® 488 goat anti-rabbit IgG (1/2000) was used as the secondary antibody. Black - Isotype control, rabbit monoclonal IgG. Blue - Unlabelled control, cells without incubation with primary and secondary antibodies. 

Immunohistochemical analysis of paraffin-embedded, Human tonsil tissue labeling Ctip1/BCL-11A with ab191401 at a 1/100 dilution (19 μg/ml). Counter stained with hematoxylin. In the negative control PBS was used instead of primary antibody.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.