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.

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.

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).

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.

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

Lane 1 : HeLa whole cell lysate - treated with Camptothecin
Lane 2 : HeLa whole cell lysate - untreated

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/1000 dilution

Predicted band size: 46 kDa
Observed band size: 35,46 kDa why is the actual band size different from the predicted?

Blocking and dilution buffer: 5% NFDM/TBST.

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.

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.

All lanes : Anti-Caspase-9 antibody [E23] (ab32539) at 1/20000 dilution (unpurified)

Lane 1 : Jurkat cell lysate - untreated
Lane 2 : Jurkat cell lysate - treated with Camptothecin

Predicted band size: 46 kDa
Observed band size: 48 kDa why is the actual band size different from the predicted?
Additional bands at: 28 kDa. We are unsure as to the identity of these extra bands.