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 - ab184787 observed at 35 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab184787 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. ab184787 and ab8245 (loading control to GAPDH) were diluted to 1/2000 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 : Anti-Caspase-3 antibody [EPR18297] (ab184787) at 0.7 µg/ml

Lane 1 : Untreated NIH/3T3 (Mouse embryonic fibroblast) whole cell lysate
Lane 2 : NIH/3T3 (Mouse embryonic fibroblast) treated with 1µM Staurosporine for 4 hours whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 32 kDa
Observed band size: 32 kDa

Blocking and diluting buffer: 5% NFDM/TBST.

ab184787 recognizes pro-Caspase 3 and unable to detect the active caspases after induction in mouse and rat samples.

All lanes : Anti-Caspase-3 antibody [EPR18297] (ab184787) at 1/2000 dilution

Lane 1 : Untreated Jurkat (Human T cell leukemia cells from peripheral blood) whole cell lysates
Lane 2 : Jurkat whole cell lysates treated with 1uM staurosporine for 4 hours

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L),Peroxidase conjugated at 1/1000 dilution

Predicted band size: 32 kDa
Observed band size: 17,32 kDa why is the actual band size different from the predicted?


Exposure time: 1 minute

Blocking/Dilution buffer: 5% NFDM/TBST.

Specificity: interacts with full length pro-Caspase 3 and the p17 subunit.

The Caspase-3 precursor is first cleaved between D175 and S176 to produce the p11 subunit and p20 fragment. Subsequently, the p20 fragment is cleaved between D28 and S29 to generate the p17 subunit (Proc. Natl. Acad. Sci. USA. 93, 7464-7469 - PMID:8755496).

All lanes : Anti-Caspase-3 antibody [EPR18297] (ab184787) at 1/2000 dilution

Lane 1 : Mouse brain tissue lysate
Lane 2 : Mouse spleen tissue lysate
Lane 3 : Rat spleen tissue lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) at 1/10000 dilution

Performed under reducing conditions.

Predicted band size: 32 kDa
Observed band size: 32 kDa

Lanes 1 - 4: Merged signal (red and green). Green - ab184787 observed at 32 kDa. Red - loading control, ab18058, observed at 130 kDa.

 

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 ab184787 and ab18058 (loading control) overnight at 4°C. Antibody binding was detected using Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) at a 1:10000 dilution for 1hr at room temperature and then imaged.

Immunohistochemical analysis of paraffin-embedded Human tonsil tissue labeling active and pro Caspase 3 with ab184787 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody at 1/500 dilution. Nucleus and cytoplasm staining on lymphocytes of tonsil is observed. Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded Human cervical cancer tissue labeling active and pro Caspase 3 with ab184787 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody at 1/500 dilution. Nucleus and cytoplasm staining on tumor cells of Human cervix cancer is observed. Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

active and pro Caspase 3 was immunoprecipitated from 1mg of HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysate treated with 1uM staurosporine for 4 hours with ab184787 at 1/80 dilution. Western blot was performed from the immunoprecipitate using ab184787 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used for detection at 1/1500 dilution.

Lane 1: HeLa whole cell lysate treated with 1uM staurosporine for 4 hours 10 µg (Input).

Lane 2: ab184787 IP in HeLa whole cell lysate treated with 1uM staurosporine for 4 hours.

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab184787 in HeLa whole cell lysate treated with 1uM staurosporine for 4 hours.
Blocking and dilution buffer and concentration: 5% NFDM/TBST.

Exposure time: 3 minutes.