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.

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 : Anti-Caspase-3 antibody [31A1067] (ab13585) at 5 µg/ml

Lane 1 : Human brain lysate
Lanes 2 & 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



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

All lanes : Anti-MDC1 antibody (ab13858) at 1 µg/ml

Lane 1 : Hela whole cell lysate (Staurosporine treated, 2uM/4hr)
Lane 2 : Hela whole cell lysate (untreated control)

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (Alexa Fluor® 790) (ab175781) at 1/10000 dilution

Performed under reducing conditions.

Predicted band size: 31 kDa
Additional bands at: 17 kDa (possible mature (processed) protein), 19 kDa (possible mature (processed) protein), 32 kDa (possible immature (unprocessed))

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.