Anti-Caspase-9 antibody [EPR18107]

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Caspase-9 antibody [EPR18107] (AB202068)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (Human epithelial cells from cervix adenocarcinoma) cells labeling Caspase-9 with ab202068 at 1/500 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/500 dilution (green).

Confocal image showing cytoplasmic and nuclear staining on HeLa cell line. The expression increased after treatment with staurosporine (1uM) for 4 hours.

The nuclear counter stain is DAPI (blue). Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution and ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution (red).

The negative controls are as follows :
-ve control 1 : ab202067 at 1/500 dilution followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution.
-ve control 2 : ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution followed by ab150077 (Alexa Fluor®488 Goat Anti-Rabbit IgG H&L) at 1/500 dilution.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Caspase-9 antibody [EPR18107] (AB202068)

Immunohistochemical analysis of paraffin-embedded Human cervix carcinoma tissue labeling Caspase-9 with ab202068 at 1/300 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody at 1/500 dilution.

Cytoplasmic and nuclear staining on Human cervix carcinoma tissue is observed.

Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• IP

Supplier Data

Immunoprecipitation - Anti-Caspase-9 antibody [EPR18107] (AB202068)

Caspase-9 was immunoprecipitated from 1mg of HeLa (Human epithelial cells from cervix adenocarcinoma) treated with staurosporine 1uM for 4 hours whole cell lysate with ab202068 at 1/80 dilution.

Western blot was performed from the immunoprecipitate using ab202068 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 treated with staurosporine 1uM for 4 hours whole cell lysate10 μg (Input).

Lane 2 : ab202068 IP in HeLa treated with staurosporine 1uM for 4 hours whole cell lysate.

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab202068 in HeLa treated with staurosporine 1uM for 4 hours whole cell lysate.
Blocking and dilution buffer and concentration : 5% NFDM/TBST.

Exposure time : 3 seconds.

All lanes:

Immunoprecipitation - Anti-Caspase-9 antibody [EPR18107] (ab202068)

Predicted band size: 46 kDa

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• WB

Lab

Western blot - Anti-Caspase-9 antibody [EPR18107] (AB202068)

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 - target observed at 46 kDa. Red - loading control, ab8245, observed at 37 kDa.

This western blot image is a comparison between ab202068 and a competitor's top cited rabbit polyclonal antibody.

All lanes:

Western blot - Anti-Caspase-9 antibody [EPR18107] (ab202068)

Predicted band size: 46 kDa

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• WB

Lab

Western blot - Anti-Caspase-9 antibody [EPR18107] (AB202068)

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 - ab202068 observed at 46 kDa. Red - loading control, ab8245, observed at 37 kDa.
ab202068 was shown to recognize Caspase-9 when Caspase-9 knockout samples were used, along with additional cross-reactive bands. Wild-type and Caspase-9 knockout samples were subjected to SDS-PAGE. ab202068 and ab8245 (loading control to GAPDH) were diluted at 1/2000 and 1/10 000 respectively and incubated overnight at 4°C. Blots were developed withGoat anti-Rabbit IgG H&L (IRDy^e® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1/10,000 dilution for 1 h at room temperature before imaging.

All lanes:

Western blot - Anti-Caspase-9 antibody [EPR18107] (ab202068)

Predicted band size: 46 kDa

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• WB

Lab

Western blot - Anti-Caspase-9 antibody [EPR18107] (AB202068)

False colour image of Western blot : Anti-Caspase-9 antibody [EPR18107] staining at 1/2000 dilution, shown in green; Mouse anti-CANX [CANX/1543] (ab238078) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab202068 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 [EPR18107] (ab202068) at 1/2000 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

Supplier Data

Western blot - Anti-Caspase-9 antibody [EPR18107] (AB202068)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-Caspase-9 antibody [EPR18107] (ab202068) at 1/2000 dilution

Lane 1:

Human fetal kidney lysate at 10 µg

Lane 2:

Human fetal liver 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: 46 kDa

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Exposure time: 30s

• WB

Supplier Data

Western blot - Anti-Caspase-9 antibody [EPR18107] (AB202068)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-Caspase-9 antibody [EPR18107] (ab202068) at 1/2000 dilution

Lane 1:

Human fetal brain lysate at 10 µg

Lane 2:

Human fetal heart 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: 46 kDa

false

Exposure time: 3min

• WB

Lab

Western blot - Anti-Caspase-9 antibody [EPR18107] (AB202068)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-Caspase-9 antibody [EPR18107] (ab202068) at 1/50000 dilution

Lane 1:

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

Lane 2:

HeLa (human epithelial cells from cervix adenocarcinoma) treated with staurosporine 1 µM for 4 hours whole cell lysate at 20 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: 46 kDa

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

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• WB

Supplier Data

Western blot - Anti-Caspase-9 antibody [EPR18107] (AB202068)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-Caspase-9 antibody [EPR18107] (ab202068) at 1/10000 dilution

Lane 1:

Untreated C2C12 (Mouse myoblast cell line) whole cell lysate at 10 µg

Lane 2:

C2C12 (Mouse myoblast cell line) treated with staurosporine 1uM for 4 hours whole cell 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,46 kDa

false

Exposure time: 3min

• WB

CiteAb

Western blot - Anti-Caspase-9 antibody [EPR18107] (AB202068)

Caspase-9 western blot using anti-Caspase-9 antibody [EPR18107] ab202068. Publication image and figure legend from Mesmar, F., Dai, B., et al., 2019, Cancer Med, PubMed 31568691.

ab202068 was used in this publication in western blot. This may not be the same as the application(s) guaranteed by Abcam. For a full list of applications guaranteed by Abcam for ab202068 please see the product overview.

GPER1 mediates anti-proliferative effects in pancreatic cancer. A, GPER1 agonist (G1, 2 μmol/L) reduces cell proliferation of both cell lines (MTT assay, 72 h), which is attenuated upon addition of GPER1 antagonist (G15). B, G1 induces PARP, caspase 3, and caspase 9 cleavage in both MiaPaCa2 and PANC1. C, Propidium iodide (PI) and Annexin V staining demonstrate apoptosis in PANC1 cells after G1 treatment (2 μmol/L, 48 h). Error bar represents SEM and unpaired two-tailed t test was used to test significance (*P < .05, **P < .01, ***P < .001)

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