All lanes : Anti-Caspase-3 antibody [E87] (ab32351) at 1/5000 dilution

Lane 1 : DMSO control wild-type HAP1 whole cell lysate
Lane 2 : Staurosporine treated wild-type HAP1 whole cell lysate
Lane 3 : DMSO control CASP3 knockout HAP1 whole cell lysate
Lane 4 : Staurosporine treated CASP3 knockout HAP1 whole cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 32 kDa

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.

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.

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.

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.

All lanes : Anti-Caspase-3 antibody [E87] (ab32351) at 1/5000 dilution (purified)

Lane 1 : untreated Jurkat (human T cell leukemia cell line from peripheral blood)cell lysate
Lane 2 : Jurkat treated with staurosporine

Lysates/proteins at 10 µg per lane.

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

Predicted band size: 32 kDa
Observed band size: 17,35 kDa why is the actual band size different from the predicted?

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

All lanes : Anti-Caspase-3 antibody [E87] (ab32351) at 1/5000 dilution (purified)

Lane 1 : Ramos (human Burkitt's lymphoma cell line) cell lysate
Lane 2 : HEK-293 (human epithelial cell line from embryonic kidney) cell lysate

Lysates/proteins at 20 µg per lane.

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

Predicted band size: 32 kDa
Observed band size: 35 kDa why is the actual band size different from the predicted?

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

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

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).

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.

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 22ºC. The secondary antibody used was Alexa Fluor^® 488 goat anti-rabbit IgG (H&L) (ab150081) at 1/2000 dilution for 30 min at 22ºC. 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.