Anti-pro Caspase-3 antibody [E61] - BSA and Azide free

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-pro Caspase-3 antibody [E61] - BSA and Azide free (AB183179)

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

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32150).

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] - BSA and Azide free (AB183179)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32150).

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-pro Caspase-3 antibody [E61] - BSA and Azide free (AB183179)

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).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32150).

• WB

Supplier Data

Western blot - Anti-pro Caspase-3 antibody [E61] - BSA and Azide free (AB183179)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (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] (<a href='/en-us/products/primary-antibodies/pro-caspase-3-antibody-e61-ab32150'>ab32150</a>) 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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• IP

Lab

Immunoprecipitation - Anti-pro Caspase-3 antibody [E61] - BSA and Azide free (AB183179)

This data was developed using ab32150, the same antibody clone in a different buffer formulation. 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] (<a href='/en-us/products/primary-antibodies/pro-caspase-3-antibody-e61-ab32150'>ab32150</a>)

Predicted band size: 31 kDa

Observed band size: 35 kDa

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• WB

Unknown

Western blot - Anti-pro Caspase-3 antibody [E61] - BSA and Azide free (AB183179)

This WB data was generated using the same anti-pro Caspase-3 antibody clone, E61, in a different buffer formulation (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] (<a href='/en-us/products/primary-antibodies/pro-caspase-3-antibody-e61-ab32150'>ab32150</a>)

Predicted band size: 31 kDa

Observed band size: 35 kDa

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• WB

CiteAb

Western blot - Anti-pro Caspase-3 antibody [E61] - BSA and Azide free (AB183179)

pro Caspase-3 western blot using anti-pro Caspase-3 antibody [E61] - BSA and Azide free ab183179. Publication image and figure legend from Gu, Y., Zhu, Z., et al., 2020, Aging (Albany NY), PubMed 32019904.

ab183179 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 ab183179 please see the product overview.

Effects of NNT-AS1 on the growth of CCLP1 and TFK1 cells, which were transfected with pcDNA-NNT-AS1 and si-NNT-AS1. (A) NNT-AS1 level was examined via qRT-PCR. (B) Proliferation was examined via BrdU. Expression of cyclinD1 was examined via western blot (C) and analyzed quantitatively (D) in both cells. (E) Apoptosis was examined via flow cytometry. Expression of cleaved-caspase-3 was examined via western blot (F) and analyzed quantitatively (G) in both cells. * p < 0.05, ** p < 0.01 and *** p < 0.001 contrasted with indicated set.

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