Anti-SATB2 antibody [EPNCIR130A]

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SATB2 antibody [EPNCIR130A] (AB92446)

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.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-SATB2 antibody [EPNCIR130A] (AB92446)

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.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-SATB2 antibody [EPNCIR130A] (AB92446)

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.

• IHC

Lab

Immunohistochemistry - Anti-SATB2 antibody [EPNCIR130A] (AB92446)

Immunohistochemical analysis of formalin fixed paraffin embedded human colon labelling SATB2 with ab92446 at a concentration of 2µg/ml. The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with a OptiView DAB IHC Detection Kit. Heat mediated antigen retrieval was performed with DISCOVERY cell conditioning solution (CC1) 100°C, pH 8.5 for 56mins.

ab92446 anti-SATB2 [EPNCIR130A] was incubated for 16mins at 37°C. Sections were counterstained with Hematoxylin II. Image inset shows absence of staining in secondary antibody only control.

Customers are encouraged to optimise antigen retrieval conditions, antibody concentration, incubation times and temperature for best results in their own IHC assay workflow (automated and manual).

• IHC

Lab

Immunohistochemistry - Anti-SATB2 antibody [EPNCIR130A] (AB92446)

Immunohistochemical analysis of formalin fixed paraffin embedded human cortex labelling SATB2 with ab92446 at a concentration of 2 µg/ml. The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with a OptiView DAB IHC Detection Kit. Heat mediated antigen retrieval was performed with DISCOVERY cell conditioning solution (CC1) 100°C, pH 8.5 for 56 mins.

ab92446 anti-SATB2 [EPNCIR130A] was incubated for 16mins at 37°C. Sections were counterstained with Hematoxylin II. Image inset shows absence of staining in secondary antibody only control.

Customers are encouraged to optimise antigen retrieval conditions, antibody concentration, incubation times and temperature for best results in their own IHC assay workflow (automated and manual)

• WB

Lab

Western blot - Anti-SATB2 antibody [EPNCIR130A] (AB92446)

Blocking buffer and concentration : 5% NFDM/TBST.

Diluting buffer and concentration : 5% NFDM /TBST.

All lanes:

Western blot - Anti-SATB2 antibody [EPNCIR130A] (ab92446) at 1/1000 dilution

All lanes:

SW1353 (human chondrosarcoma cell line) cell lysate at 20 µg

Secondary

All lanes:

Peroxidase conjugated goat anti-rabbit IgG (H+L) at 1/1000 dilution

Predicted band size: 83 kDa

Observed band size: 83 kDa

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• WB

Lab

Western blot - Anti-SATB2 antibody [EPNCIR130A] (AB92446)

Blocking buffer and concentration : 5% NFDM/TBST.

Diluting buffer and concentration : 5% NFDM /TBST.

All lanes:

Western blot - Anti-SATB2 antibody [EPNCIR130A] (ab92446) at 1/1000 dilution

Lane 1:

Saos-2 (human osteosarcoma cell line) cell lysate at 20 µg

Lane 2:

Human fetal brain tissue lysate at 20 µg

Secondary

All lanes:

Peroxidase conjugated goat anti-rabbit IgG (H+L) at 1/1000 dilution

Predicted band size: 83 kDa

Observed band size: 83 kDa

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• WB

Lab

Western blot - Anti-SATB2 antibody [EPNCIR130A] (AB92446)

Blocking and diluting buffer and concentration : 5% NFDM/TBST

Freshly made lysate is highly recommended to minimize protein degradation.

All lanes:

Western blot - Anti-SATB2 antibody [EPNCIR130A] (ab92446) at 1/10000 dilution

Lane 1:

Saos-2 (Human osteosarcoma epithelial) whole cell fresh lysate at 10 µg

Lane 2:

Saos-2 (Human osteosarcoma epithelial) whole cell lysate at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 83 kDa

Observed band size: 83 kDa

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Exposure time: 20s

• WB

Lab

Western blot - Anti-SATB2 antibody [EPNCIR130A] (AB92446)

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.

All lanes:

Western blot - Anti-SATB2 antibody [EPNCIR130A] (ab92446) at 1/1000 dilution

Lane 1:

Wild-type HAP1 whole cell lysate at 20 µg

Lane 2:

SATB2 knockout HAP1 whole cell lysate at 20 µg

Predicted band size: 83 kDa

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• WB

Unknown

Western blot - Anti-SATB2 antibody [EPNCIR130A] (AB92446)

All lanes:

Western blot - Anti-SATB2 antibody [EPNCIR130A] (ab92446) at 1/1000 dilution

Lane 1:

HT1080 (human fibrosarcoma cell line) cell lysate at 10 µg

Lane 2:

SW1353 (human chondrosarcoma cell line) cell lysate at 10 µg

Lane 3:

MCF7 (human breast adenocarcinoma cell line) cell lysate at 10 µg

Secondary

All lanes:

HRP labelled goat anti-rabbit at 1/2000 dilution

Predicted band size: 83 kDa

false

• WB

Lab

Western blot - Anti-SATB2 antibody [EPNCIR130A] (AB92446)

Blocking buffer and concentration : 5% NFDM/TBST.

Diluting buffer and concentration : 5% NFDM /TBST.

All lanes:

Western blot - Anti-SATB2 antibody [EPNCIR130A] (ab92446) at 1/1000 dilution

Lane 1:

Mouse brain tissue lysate at 20 µg

Lane 2:

Rat brain tissue lysate at 20 µg

Secondary

All lanes:

Peroxidase conjugated goat anti-rabbit IgG (H+L) at 1/1000 dilution

Predicted band size: 83 kDa

Observed band size: 83 kDa

false

• WB

Lab

Western blot - 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.

Lanes 1 - 10:

Western blot - Anti-SATB2 antibody [EPNCIR130A] (ab92446) at 1/1000 dilution

Lanes 1 - 10:

Western blot - Anti-SATB2 antibody [EPNCIR130A] - BSA and Azide free (<a href='/en-us/products/primary-antibodies/satb2-antibody-epncir130a-bsa-and-azide-free-ab212177'>ab212177</a>) at 1/1000 dilution

Lane 1:

Wild-type HAP1 cell lysate at 20 µg

Lane 2:

SATB2 knockout HAP1 cell lysate at 20 µg

Lane 3:

Mouse E18 Embyonic brain cell lysate at 20 µg

Lane 4:

NIH/3T3 cell lysate at 20 µg

Lane 5:

HT1080 cell lysate at 20 µg

Lane 6:

Saos-2 cell lysate at 20 µg

Lanes 7 and 9:

Empty

Lane 8:

SATB1 Recombinant Protein cell lysate at 0.1 µg

Lane 10:

SATB2 Recombinant Protein (<a href='/en-us/products/proteins-peptides/recombinant-human-satb2-protein-ab132405'>ab132405</a>) cell lysate at 0.1 µg

Secondary

All lanes:

Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

Observed band size: 100 kDa

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