Recombinant Anti-SATB2 antibody [EPNCIR130A] (ab92446)

False colour image of Western blot: Anti-SATB2 antibody [EPNCIR130A] staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab92446 was shown to bind specifically to SATB2. A band was observed at 100 kDa in wild-type HAP1 cell lysates with no signal observed at this size in SATB2 knockout cell line.

To generate this image, wild-type and SATB2 knockout HAP1 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

Immunocytochemistry/Immunofluorescence analysis of Saos-2 cells (human osteosarcoma cell line) labelling SATB2 (red) with purified ab92446 at 1/100 dilution. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. ab150083, an Alexa Fluor® 647-conjugated goat anti-rabbit IgG H&L (1/1000), was used as the secondary antibody. Tubulin (green) was stained with ab7291, an anti-alpha tubulin antibody (1/1000). DAPI (blue) was used as the nuclear counterstain.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human cerebral cortex tissue labelling SATB2 with purified ab92446 at 1/150. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. A prediluted HRP-polymer conjugated anti-rabbit IgG was used as the secondary antibody. Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

Lanes 1 - 2: Merged signal (red and green). Green - ab92446 observed at 83 kDa. Red - loading control, ab9484, observed at 37 kDa.

ab92446 was shown to specifically react with SATB2 in wild-type HAP1 cells as signal was lost in SATB2 knockout cells. Wild-type and SATB2 knockout samples were subjected to SDS-PAGE. Ab92446 and ab9484 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1/1000 dilution and 1/20000 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 1/20000 dilution for 1 hour at room temperature before imaging.

Intracellular Flow Cytometry analysis of SH-SY5Y cells (human neuroblastoma cell line from bone marrow) labeling SATB2 with purified ab92446 at 1/150 dilution (10ug/ml) (red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% Methanol. A Goat anti rabbit IgG (Alexa Fluor^® 488) (ab150077) (1/2000 dilution) was used as the secondary antibody. Rabbit monoclonal IgG (Black) (ab172730) was used as the isotype control, cells without incubation with primary antibody and secondary antibody (Blue) were used as the unlabeled control. 

Blocking buffer and concentration: 5% NFDM/TBST.

Diluting buffer and concentration: 5% NFDM /TBST.

Blocking buffer and concentration: 5% NFDM/TBST.

Diluting buffer and concentration: 5% NFDM /TBST.

Blocking buffer and concentration: 5% NFDM/TBST.

Diluting buffer and concentration: 5% NFDM /TBST.