Lane 1: Wild-type HAP1 cell lysate (20 µg)
Lane 2: Caspase-9 knockout HAP1 cell lysate (20 µg)
Lane 3: HeLa cell lysate (20 µg)
Lane 4: Jurkat cell lysate (20 µg)
Lanes 1 - 4: Merged signal (red and green). Green - ab185719 observed at 46 kDa. Red - loading control, ab8245, observed at 37 kDa.
ab185719 was shown to specifically react with Caspase-9 when Caspase-9 knockout samples were used. Wild-type and Caspase-9 knockout samples were subjected to SDS-PAGE. ab185719 and ab8245 (loading control to GAPDH) were diluted at 1/1000 and 1/10000 respectively and incubated overnight at 4°C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1/10000 dilution for 1 h at room temperature before imaging.

All lanes : Anti-Caspase-9 antibody [EPR18108] (ab185719) at 1/10000 dilution

Lane 1 : Untreated HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysate
Lane 2 : HeLa whole cell lysate treated with staurosporine 1uM for 4 hours

Lysates/proteins at 10 µg per lane.

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

Predicted band size: 46 kDa
Observed band size: 35,37,46 kDa why is the actual band size different from the predicted?


Exposure time: 10 seconds

Blocking and dilution buffer: 5% NFDM/TBST.

All lanes : Anti-Caspase-9 antibody [EPR18108] (ab185719) at 1/2500 dilution

Lane 1 : Untreated NIH/3T3 (Mouse embyro fibroblast cells) whole cell lysate
Lane 2 : NIH/3T3 whole cell lysate treated with staurosporine 1uM for 4 hours

Lysates/proteins at 10 µg per lane.

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

Predicted band size: 46 kDa
Observed band size: 37,39,49 kDa why is the actual band size different from the predicted?


Exposure time: 30 seconds

Blocking and dilution buffer: 5% NFDM/TBST.

All lanes : Anti-Caspase-9 antibody [EPR18108] (ab185719) at 1/1000 dilution

Lane 1 : Human fetal brain lysate
Lane 2 : Human fetal heart lysate
Lane 3 : Human fetal kidney lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/1000 dilution

Predicted band size: 46 kDa
Observed band size: 46 kDa


Exposure time: 30 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.

All lanes : Anti-Caspase-9 antibody [EPR18108] (ab185719) at 1/5000 dilution

Lane 1 : Human pancreas lysate
Lane 2 : Human fetal liver lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/1000 dilution

Predicted band size: 46 kDa
Observed band size: 46 kDa


Exposure time: 30 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.

All lanes : Anti-Caspase-9 antibody [EPR18108] (ab185719) at 1/1000 dilution

Lane 1 : Mouse heart lysate
Lane 2 : Mouse kidney lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L),Peroxidase conjugated at 1/1000 dilution

Predicted band size: 46 kDa
Observed band size: 37,39,49 kDa why is the actual band size different from the predicted?


Exposure time: 15 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.

Caspase-9 was immunoprecipitated from 1mg of HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysate treated with staurosporine 1uM for 4 hours with ab185719 at 1/40 dilution. Western blot was performed from the immunoprecipitate using ab185719 at 1/1000 dilution. Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG, was used as secondary antibody at 1/1500 dilution.

Lane 1: HeLa whole cell lysate treated with staurosporine 10 µg (Input).

Lane 2: ab185719 IP in HeLa whole cell lysate treated with staurosporine.

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab185719 in HeLa whole cell lysate treated with staurosporine.
Blocking and dilution buffer and concentration: 5% NFDM/TBST.