Anti-Caspase-3 antibody [31A1067]

• WB

Unknown

Western blot - Anti-Caspase-3 antibody [31A1067] (AB13585)

This blot was produced using a 4-12% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using Licor blocking buffer before being incubated with ab13585 overnight at 4°C. Antibody binding was detected using ab175781 at a 1 : 10,000 dilution for 1hr at room temperature and then imaged using the Licor Odyssey CLx.

All lanes:

Anti-MDC1 antibody (<a href='/ko/products/unavailable/mdc1-antibody-ab13858'>ab13858</a>) at 1 µg/mL

Lane 1:

Hela whole cell lysate (Staurosporine treated, 2uM/4hr) at 20 µg

Lane 2:

Hela whole cell lysate (untreated control) at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (Alexa Fluor® 790) (<a href='/ko/products/secondary-antibodies/goat-rabbit-igg-h-l-alexa-fluor-790-ab175781'>ab175781</a>) at 1/10000 dilution

Predicted band size: 22 kDa,28 kDa,29 kDa,31 kDa,50 kDa

Observed band size: 17 kDa,19 kDa,32 kDa,36 kDa,43 kDa

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• WB

Unknown

Western blot - Anti-Caspase-3 antibody [31A1067] (AB13585)

Western blot analysis of Caspase 3 in HeLa cells using ab13585. Cells were treated with 2 uM staurosporine for different time periods. Caspase 3 activation is detected in Western blots by the presence of Caspase 3 cleavage fragments.

ab13585 detected both pro (full-length) and active (cleaved) Caspase 3, depending on the treatment time points. Pro Caspase 3 is detected at ~32 kDa. Active/cleaved Caspase 3 (large subunit) is detected at ~14-21 kDa as one or more bands.

All lanes:

Western blot - Anti-Caspase-3 antibody [31A1067] (ab13585)

Predicted band size: 31 kDa

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• WB

Lab

Western blot - Anti-Caspase-3 antibody [31A1067] (AB13585)

Lane 1 : Wild-type HAP1 cell lysate + Staurosporine (1μM for 4h)
Lane 2 : Wild-type HAP1 cell lysate
Lane 3 : Caspase-3 knockout HAP1 cell lysate + Staurosporine (1μM for 4h)
Lane 4 : Caspase-3 knockout HAP1 cell lysate
Lanes 1 - 4 : Merged signal (red and green). Green - ab13585 observed at 32 kDa. Red - loading control, ab181602, observed at 37 kDa.

ab13585 was shown to recognise pro Caspase 3 when Caspase 3 knockout samples were used, along with additional cross-reactive bands. Wild-type and Caspase 3 knockout samples (± Staurosporine treatment) were subjected to SDS-PAGE. ab13585 at a concentration of 1 μg/ml and ab8245 (loading control to GAPDH) diluted to 1/10000 were incubated overnight at 4°C. Blots were developed with Goat anti-Mouse IgG H&L (IRDye^® 800CW) preadsorbed (ab216772) and Goat Anti-Rabbit IgG H&L (IRDye^® 680RD) preadsorbed (ab216777) secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-Caspase-3 antibody [31A1067] (ab13585)

Predicted band size: 31 kDa

Observed band size: 32 kDa

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• WB

Unknown

Western blot - Anti-Caspase-3 antibody [31A1067] (AB13585)

Lanes 12, 13 and 14 demonstrate the species cross-reactivity of the antibody in Human, mouse and rat heart lysate, respectively.

All lanes:

Western blot - Anti-Caspase-3 antibody [31A1067] (ab13585) at 5 µg/mL

Lane 1:

Human brain lysate

Lanes 2 and 12:

Human heart lysate

Lane 3:

Human intestine lysate

Lane 4:

Human kidney lysate

Lane 5:

Human liver lysate

Lane 6:

Human lung lysate

Lane 7:

Human muscle lysate

Lane 8:

Human stomach lysate

Lane 9:

Human spleen lysate

Lane 10:

Human ovary lysate

Lane 11:

Human testis lysate

Lane 13:

Mouse heart lysate

Lane 14:

Rat heart lysate

Predicted band size: 31 kDa

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