Anti-pro Caspase-3 抗体 [E61]

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-pro Caspase-3 antibody [E61] (AB32150)

Immunohistochemical analysis of human paraffin-embedded cervical carcinoma tissue using ab32150 at 1/500 dilution.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-pro Caspase-3 antibody [E61] (AB32150)

Overlay histogram showing Jurkat cells stained with ab32150 (red line). The cells were fixed with 80% 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. The cells were then incubated with the antibody (ab32150, 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 IgG (1μg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in Jurkat cells fixed with 4% paraformaldehyde (10 min)/permeabilized in 0.1% PBS-Tween used under the same conditions.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-pro Caspase-3 antibody [E61] (AB32150)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized Jurkat (Human T cell leukemia cell line from peripheral blood) cells labelling pro Caspase-3 with primary antibody anti-pro Caspase-3 (ab32150) at 1/200 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150081) secondary antibody at 1/1000 dilution. Confocal image showing cytoplasmic staining in Jurkat cells. Anti-alpha Tubulin antibody (DM1A) - Microtubule Marker (Alexa Fluor^® 594) (ab195889) was used to counterstain tubulin at 1/200 dilution. The nuclear counter stain is DAPI (blue). Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

• IP

Lab

Immunoprecipitation - Anti-pro Caspase-3 antibody [E61] (AB32150)

pro Caspase-3 was immunoprecipitated from 0.35 mg HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate 10 μg with ab32150 at 1/20 dilution (0.6μg). VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1/5000 dilution.

Lane 1 : HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate 10 μg

Lane 2 : ab32150 IP in HeLa whole cell lysate

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab32150 in HeLa whole cell lysate

Blocking and dilution buffer and concentration : 5% NFDM/TBST.

All lanes:

Immunoprecipitation - Anti-pro Caspase-3 antibody [E61] (ab32150)

Predicted band size: 31 kDa

Observed band size: 35 kDa

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• WB

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Western blot - Anti-pro Caspase-3 antibody [E61] (AB32150)

Lane 1 : Wild-type HAP1 cell lysate
Lane 2 : Wild-type HAP1 cell lysate + Staurosporine (1μM for 4h)
Lane 3 : Caspase-3 knockout HAP1 cell lysate
Lane 4 : Caspase-3 knockout HAP1 cell lysate + Staurosporine (1μM for 4h)
Lanes 1 - 4 : Merged signal (red and green). Green - ab32150 observed at 35 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab32150 was shown to specifically react with pro Caspase 3 when Caspase 3 knockout samples were used. Wild-type and Caspase 3 knockout samples (± Staurosporine treatment) were subjected to SDS-PAGE. ab32150 and ab8245 (loading control to GAPDH) were diluted to 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 hour at room temperature before imaging.

All lanes:

Western blot - Anti-pro Caspase-3 antibody [E61] (ab32150)

Predicted band size: 31 kDa

Observed band size: 35 kDa

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• WB

Supplier Data

Western blot - Anti-pro Caspase-3 antibody [E61] (AB32150)

Anti-CASP3 antibody [E61] (ab32150) staining at 1/1000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab32150 was shown to bind specifically to CASP3. A band was observed at 30 kDa in treated wild-type HeLa cell lysates with no signal observed at this size in CASP3 knockout cell line. To generate this image, wild-type and CASP3 knockout HeLa 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 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-pro Caspase-3 antibody [E61] (ab32150) at 1/500 dilution

Lane 1:

Wild-type HeLa Treated Staurosporine (2uM, 4h) cell lysate at 20 µg

Lane 2:

CASP3 knockout HeLa Treated Staurosporine (2uM, 4h) cell lysate at 20 µg

Lane 3:

Wild-type HeLa Vehicle Control Staurosporine (0uM, 4h) cell lysate at 20 µg

Lane 4:

CASP3 knockout HeLa Vehicle Control Staurosporine (0uM, 4h) cell lysate at 20 µg

Secondary

Lanes 1 - 4:

Goat anti-Rabbit IgG H&L 800CW at 1/20000 dilution

Lanes 1 - 4:

Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

Predicted band size: 31 kDa

Observed band size: 30 kDa

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• WB

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Western blot - Anti-pro Caspase-3 antibody [E61] (AB32150)

All lanes:

Western blot - Anti-pro Caspase-3 antibody [E61] (ab32150) at 1/1000 dilution

All lanes:

Hela cell lysate

Predicted band size: 31 kDa

Observed band size: 35 kDa

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