Anti-Caspase-9 antibody [EPR18108]

• IP

Supplier Data

Immunoprecipitation - Anti-Caspase-9 antibody [EPR18108] (AB185719)

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.

All lanes:

Immunoprecipitation - Anti-Caspase-9 antibody [EPR18108] (ab185719)

Predicted band size: 46 kDa

false

• WB

Lab

Western blot - Anti-Caspase-9 antibody [EPR18108] (AB185719)

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:

Western blot - Anti-Caspase-9 antibody [EPR18108] (ab185719)

Predicted band size: 46 kDa

false

• WB

Lab

Western blot - Anti-Caspase-9 antibody [EPR18108] (AB185719)

False colour image of Western blot : Anti-Caspase-9 antibody [EPR18108] 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, ab185719 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 [EPR18108] (ab185719) 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

false

• WB

Supplier Data

Western blot - Anti-Caspase-9 antibody [EPR18108] (AB185719)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-Caspase-9 antibody [EPR18108] (ab185719) at 1/1000 dilution

Lane 1:

Human fetal brain lysate at 10 µg

Lane 2:

Human fetal heart lysate at 10 µg

Lane 3:

Human fetal kidney lysate at 10 µg

Secondary

All lanes:

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

Predicted band size: 46 kDa,82 kDa

Observed band size: 46 kDa

false

Exposure time: 30s

• WB

Lab

Western blot - Anti-Caspase-9 antibody [EPR18108] (AB185719)

Blocking and dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-Caspase-9 antibody [EPR18108] (ab185719) at 1/10000 dilution

Lane 1:

Untreated HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysate at 10 µg

Lane 2:

HeLa whole cell lysate treated with staurosporine 1uM for 4 hours at 10 µg

Secondary

All lanes:

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

Predicted band size: 46 kDa

Observed band size: 35 kDa,37 kDa,46 kDa

false

Exposure time: 10s

• WB

Supplier Data

Western blot - Anti-Caspase-9 antibody [EPR18108] (AB185719)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-Caspase-9 antibody [EPR18108] (ab185719) at 1/5000 dilution

Lane 1:

Human pancreas tissue lysate at 10 µg

Lane 2:

Human fetal liver tissue lysate at 10 µg

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

false

Exposure time: 30s

• WB

Supplier Data

Western blot - Anti-Caspase-9 antibody [EPR18108] (AB185719)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-Caspase-9 antibody [EPR18108] (ab185719) at 1/1000 dilution

Lane 1:

Mouse heart tissue lysate at 10 µg

Lane 2:

Mouse kidney tissue lysate at 10 µg

Secondary

All lanes:

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

Predicted band size: 46 kDa

Observed band size: 37 kDa,39 kDa,49 kDa

false

Exposure time: 15s

• WB

Lab

Western blot - Anti-Caspase-9 antibody [EPR18108] (AB185719)

Blocking and dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-Caspase-9 antibody [EPR18108] (ab185719) at 1/2500 dilution

Lane 1:

Untreated NIH/3T3 (Mouse embyro fibroblast cells) whole cell lysate at 10 µg

Lane 2:

NIH/3T3 whole cell lysate treated with staurosporine 1uM for 4 hours at 10 µg

Secondary

All lanes:

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

Predicted band size: 46 kDa

Observed band size: 37 kDa,39 kDa,49 kDa

false

Exposure time: 30s