Anti-Caspase-3 抗体 [E87]

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Caspase-3 antibody [E87] (AB32351)

Unpurified ab32351, at a 1/25 dilution, staining Capase-3 in paraffin embedded human cervical carcinoma tissue by Immunohistochemistry.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-Caspase-3 antibody [E87] (AB32351)

ab32351 staining Caspase-3 in wild-type Hap1 cells (top panel) and CASP3 knockout Hap1 cells (bottom panel). The cells were fixed with 100% methanol (5 min) then permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab32351 at 1μg/ml concentration and ab7291 (Mouse monoclonal to alpha Tubulin) at 1/1000 dilution overnight at 4°C followed by a further incubation at room temperature for 1h with a goat secondary antibody to rabbit IgG (Alexa Fluor® 488) (ab150081) at 2 μg/ml (shown in green) and a goat secondary antibody to mouse IgG (Alexa Fluor® 594) (ab150120) at 2 μg/ml (shown in red). Nuclear DNA was labelled in blue with DAPI. Image was taken with a confocal microscope (Leica-Microsystems TCS SP8).

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Caspase-3 antibody [E87] (AB32351)

Immunohistochemical analysis of formalin fixed paraffin embedded human tonsil labelling caspase-3 with ab32351 at a concentration of 0.1µg/ml. The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with a OptiView DAB IHC Detection Kit. Heat mediated antigen retrieval was performed with DISCOVERY cell conditioning solution (CC1) 100°C, pH8.5 for 32mins.

ab32351 anti-Caspase-3 antibody [E87] was incubated for 16mins at 37°C. Sections were counterstained with Hematoxylin II. Image inset shows absence of staining in secondary antibody only control.

Customers are encouraged to optimise antigen retrieval conditions, antibody concentration, incubation times and temperature for best results in their own IHC assay workflow (automated and manual)

• WB

Lab

Western blot - Anti-Caspase-3 antibody [E87] (AB32351)

Blocking buffer : 5% NFDM/TBST
Dilution buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-Caspase-3 antibody [E87] (ab32351) at 1/5000 dilution

Lane 1:

Ramos (human Burkitt's lymphoma cell line) cell lysate at 20 µg

Lane 2:

HEK-293 (human epithelial cell line from embryonic kidney) cell lysate at 20 µg

Secondary

All lanes:

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

Predicted band size: 31 kDa

Observed band size: 35 kDa

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• WB

Lab

Western blot - Anti-Caspase-3 antibody [E87] (AB32351)

Lanes 1 - 4 : Merged signal (red and green). Green - ab32351 observed at 31 kDa. Red - loading control, ab130007, observed at 130 kDa.

ab32351 was shown to recognize Caspase 3 in wild-type HAP1 cells as signal was lost at the expected MW in HAP1 Staurosporine Treated (CASP3) knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and HAP1 Staurosporine Treated (CASP3) knockout samples were subjected to SDS-PAGE. ab32351 and ab130007 (Mouse anti-Vinculin loading control) were incubated overnight at 4°C at 1/5000 dilution and 1/20000 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 1/20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-Caspase-3 antibody [E87] (ab32351) at 1/5000 dilution

Lane 1:

DMSO control wild-type HAP1 whole cell lysate at 20 µg

Lane 2:

Staurosporine treated wild-type HAP1 whole cell lysate at 20 µg

Lane 3:

DMSO control CASP3 knockout HAP1 whole cell lysate at 20 µg

Lane 4:

Staurosporine treated CASP3 knockout HAP1 whole cell lysate at 20 µg

Predicted band size: 31 kDa

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• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Caspase-3 antibody [E87] (AB32351)

Immunofluorescence staining of Jurkat (human T cell leukemia cell line from peripheral blood) cells with purified ab32351 at a working dilution of 1/500, counter-stained with DAPI. The secondary antibody was Alexa Fluor^® 488 goat anti-rabbit (ab150077), used at a dilution of 1/1000. ab7291, a mouse anti-tubulin antibody (1/1000), was used to stain tubulin along with ab150120 (Alexa Fluor^® 594 goat anti-mouse, 1/1000), shown in the top right hand panel. The cells were fixed in 4% PFA and permeabilized using 0.1% Triton X 100. The negative controls are shown in bottom middle and right hand panels - for negative control 1, purified ab32351 was used at a dilution of 1/500 followed by an Alexa Fluor^® 594 goat anti-mouse antibody (ab150120) at a dilution of 1/500. For negative control 2, ab7291 (mouse anti-tubulin) was used at a dilution of 1/500 followed by an Alexa Fluor^® 488 goat anti-rabbit antibody (ab150077) at a dilution of 1/400.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Caspase-3 antibody [E87] (AB32351)

Immunohistochemical staining of paraffin embedded human tonsil with purified ab32351 at a working dilution of 1/100. The secondary antibody used is HRP goat anti-rabbit IgG H&L (ab97051) at 1/500. The sample is counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0. PBS was used instead of the primary antibody as the negative control, and is shown in the inset.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Caspase-3 antibody [E87] (AB32351)

Overlay histogram showing Ramos (human Burkitt's lymphoma cell line) cells fixed in 4% PFA and stained with purified ab32351 at a dilution of 1 in 180 (red line). The secondary antibody used was FITC goat anti-rabbit at a dilution of 1 in 500. Rabbit monoclonal IgG was used as an isotype control (black line) and cells incubated in the absence of both primary and secondary antibody were used as a negative control (blue line).

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Caspase-3 antibody [E87] (AB32351)

Overlay histogram showing HeLa (human epithelial cell line from cervix adenocarcinoma) cells stained with unpurfied ab32351 (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 followed by the antibody (ab32351, 1/1000 dilution) for 30 min at 22°C. The secondary antibody used was Alexa Fluorr^®488 goat anti-rabbit IgG (H&L) (ab150081) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (ab172730, 0.1μg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control.

Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.

• IP

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Immunoprecipitation - Anti-Caspase-3 antibody [E87] (AB32351)

ab32351 (purified) at 1/50 immunoprecipitating Cullin 1 in 10 μg HeLa (human epithelial cell line from cervix adenocarcinoma) whole cell lysate (Lanes 1 and 2, observed at 35 kDa). Lane 3 - PBS. For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366) was used for detection (1/1500). Blocking buffer and concentration : 5% NFDM/TBST Dilution buffer and concentration : 5% NFDM/TBST

All lanes:

Immunoprecipitation - Anti-Caspase-3 antibody [E87] (ab32351)

Predicted band size: 31 kDa

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• WB

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Western blot - Anti-Caspase-3 antibody [E87] (AB32351)

Carried out with unpurified antibody. Lane 1 = Caspase 3 protein (Active) (ab52314) 20 ng. Lane 2 = Caspase 9 protein (Active) (ab52203) 20 ng. Lane 3 = Extract of HeLa (human epithelial cell line from cervix adenocarcinoma) cells treated with vehicle (ab136806) 20 ug. Lane 4 = Extract of HeLa cells treated with staurosporine (ab136806) 20 ug. SDS PAGE performed under reducing conditions (100 mM DTT Sample heated at 50°C). Primary : Lanes 1-4 : Anti Caspase 3 antibody (ab32351) at 1 : 1000 dilution. Secondary : Lanes 1-4 : Goat anti rabbit IgG(H&L)-HRP at 1 : 10000. Development : ECL for 10 min exposure. Blocking : in 5% Milk + PBS overnight at 4 C. Primary antibody : in 5% Milk + PBS for 2 hours at RT. Secondary antibody : in 5% Milk + PBS for 2 hours at RT. Predicted band size : 32 kDa and 17 kDa. Observed band size : 32 kDa and 17 kDa.

All lanes:

Western blot - Anti-Caspase-3 antibody [E87] (ab32351)

Predicted band size: 31 kDa

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• WB

Lab

Western blot - Anti-Caspase-3 antibody [E87] (AB32351)

Blocking buffer : 5% NFDM/TBST
Dilution buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-Caspase-3 antibody [E87] (ab32351) at 1/5000 dilution

Lane 1:

untreated Jurkat (human T cell leukemia cell line from peripheral blood)cell lysate at 10 µg

Lane 2:

Jurkat treated with staurosporine at 10 µg

Secondary

All lanes:

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

Predicted band size: 31 kDa

Observed band size: 17 kDa,35 kDa

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