Anti-Caspase-9 antibody [E23]

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Caspase-9 antibody [E23] (AB32539)

Overlay histogram showing K562 cells stained with unpurified ab32539 (red line). The cells were fixed with 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 (unpurified ab32539, 1/100 dilution) for 30 min at 22°C. The secondary antibody used was anti-rabbit DyLight^® 488 (ab96899) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit monoclonal IgG (1μg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in K562 cells fixed with 4% paraformaldehyde (10 min)/permeabilized with 0.1% PBS-Tween used under the same conditions.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Caspase-9 antibody [E23] (AB32539)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human cervical carcinoma tissue labelling Caspase 9 with unpurified ab32539 at a dilution of 1/50.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Caspase-9 antibody [E23] (AB32539)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human skeletal muscle tissue labelling Caspase-9 with purified ab32539 at 1/250. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Caspase-9 antibody [E23] (AB32539)

Intracellular Flow Cytometry analysis of K562 cells labelling Caspase-9 with purified ab32539 at 1/250 (red). Cells were fixed with 100% methanol. A FITC-conjugated goat anti-rabbit IgG (1/500) was used as the secondary antibody. Black - Isotype control, rabbit monoclonal IgG. Blue - Unlabelled control, cells without incubation with primary and secondary antibodies.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Caspase-9 antibody [E23] (AB32539)

Immunocytochemistry/Immunofluorescence analysis of HepG2 cells labelling Caspase-9 with purified ab32539 at 1/500. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody. DAPI (blue) was used as the nuclear counterstain. ab7291, a mouse anti-tubulin (1/1000) and ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/1000) were also used.

Control 1 : primary antibody (1/500) and secondary antibody, ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/1000).

Control 2 : ab7291 (1/1000) and secondary antibody, ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000).

• IP

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Immunoprecipitation - Anti-Caspase-9 antibody [E23] (AB32539)

ab32539 (purified) at 1/80 immunoprecipitating Caspase-9 in HeLa whole cell lysate.

Lane 1 (input) : HeLa whole cell lysate (10μg)

Lane 2 (+) : ab32539 + HeLa whole cell lysate.

Lane 3 (-) : Rabbit monoclonal IgG (ab172730) instead of ab32539 in HeLa whole cell lysate.

For western blotting, a HRP-conjugated anti-rabbit IgG, specific to the non-reduced form of IgG was used as the secondary antibody (1/1500).

Blocking buffer and concentration : 5% NFDM/TBST.

Diluting buffer and concentration : 5% NFDM /TBST.

All lanes:

Immunoprecipitation - Anti-Caspase-9 antibody [E23] (ab32539)

Predicted band size: 46 kDa

Observed band size: 46 kDa

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• WB

Lab

Western blot - Anti-Caspase-9 antibody [E23] (AB32539)

Blocking and dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-Caspase-9 antibody [E23] (ab32539) at 1/1000 dilution

Lane 1:

HeLa whole cell lysate - treated with Camptothecin at 10 µg

Lane 2:

HeLa whole cell lysate - untreated 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 1/1000 dilution

Predicted band size: 46 kDa

Observed band size: 35 kDa,46 kDa

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• WB

Lab

Western blot - Anti-Caspase-9 antibody [E23] (AB32539)

False colour image of Western blot : Anti-Caspase-9 antibody [E23] staining at 1/1000 dilution, shown in green; Mouse anti-CANX [CANX/1543] (ab238078) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab32539 was shown to bind specifically to Caspase-9. A band was observed at 45 kDa in wild-type THP-1 cell lysates with no signal observed at this size in CASP9 knockout cell line ab276122 (knockout cell lysate ab284219). To generate this image, wild-type and CASP9 knockout THP-1 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 (IRDye® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) at 1/20000 dilution.

All lanes:

Western blot - Anti-Caspase-9 antibody [E23] (ab32539) at 1/1000 dilution

Lane 1:

Wild-type THP-1 cell lysate at 20 µg

Lane 2:

CASP9 knockout THP-1 cell lysate at 20 µg

Lane 2:

Western blot - Human CASP9 knockout THP-1 cell line (<a href='/en-us/products/cell-lines/human-casp9-knockout-thp-1-cell-line-ab276122'>ab276122</a>)

Lane 3:

HeLa cell lysate at 20 µg

Predicted band size: 46 kDa

Observed band size: 45 kDa

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• WB

Lab

Western blot - Anti-Caspase-9 antibody [E23] (AB32539)

Lanes :
Lane 1 : Wild-type HAP1 whole cell lysate (40 μg)
Lane 2 : CASP9 knockout HAP1 whole cell lysate (40 μg)
Lane 3 : HeLa whole cell lysate (40 μg)

Lanes 1 - 4 : Merged signal (red and green). Green - ab32539 observed at 45 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab32539 was shown to specifically recognize Caspase-9 in wild type HAP1 cells along with additional cross-reactive bands. No band was observed when Caspase-9 knockout samples were examined. Wild-type and Caspase-9 knockout samples were subjected to SDS-PAGE. ab32539 and ab8245 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at 1/500 dilution and 1/10,000 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 1/10,000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-Caspase-9 antibody [E23] (ab32539)

Predicted band size: 46 kDa

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• WB

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Western blot - Anti-Caspase-9 antibody [E23] (AB32539)

All lanes:

Western blot - Anti-Caspase-9 antibody [E23] (ab32539) at 1/20000 dilution

Lane 1:

Jurkat cell lysate - untreated

Lane 2:

Jurkat cell lysate - treated with Camptothecin

Predicted band size: 46 kDa

Observed band size: 28 kDa,48 kDa

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