All lanes : Anti-Caspase-7 antibody [EPR22839-12] (ab255818) at 1/1000 dilution

Lane 1 : Untreated HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate
Lane 2 : HeLa treated with 1 µM staurosporine for 3 hours whole cell lysate
Lane 3 : C2C12 (mouse myoblasts myoblast) 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: 34 kDa
Observed band size: 18,32,35 kDa why is the actual band size different from the predicted?

Caspase-7 is activated upon apoptotic induction. Procapase-7 (35 kDa), intermediate caspase-7 (32 kDa) and caspase-7 p20 subunit (18 kDa) are observed. The molecular weight observed is consistent with what has been described in the literature (PMID: 15953353, 18596415).

Exposure time: Lanes 1-2: 15 seconds; Lane 3: 3 seconds.

Blocking and Diluting Buffer and concentration: 5% NFDM/TBST.

All lanes : Anti-Caspase-7 antibody [EPR22839-12] (ab255818) at 1/1000 dilution

Lane 1 : Untreated wild-type HAP1 whole cell lysate
Lane 2 : Wild-type HAP1 treated with 1 µM staurosporine for 3 hours whole cell lysate
Lane 3 : Untreated caspase-7 knockout HAP1 whole cell lysate
Lane 4 : Caspase-7 knockout HAP1 treated with 1 µM staurosporine for 3 hours whole cell lysate
Lane 5 : C6 (rat glial tumor glial cell) whole cell lysate
Lane 6 : PC-12 (rat adrenal gland pheochromocytoma) whole cell lysate
Lane 7 : RAW 264.7 (mouse abelson murine leukemia virus-induced tumor macrophage) 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: 34 kDa
Observed band size: 32,35 kDa why is the actual band size different from the predicted?


Exposure time: 26 seconds

Caspase-7 is activated upon apoptotic induction. Procapase-7 (35 kDa), intermediate caspase-7 (32 kDa) and caspase-7 p20 subunit (18 kDa) are observed. The molecular weight observed is consistent with what has been described in the literature (PMID: 15953353, 18596415). 

ab255818 was shown to specifically react with Caspase-7 in wild-type HAP1 cells as signal was lost in Caspase-7 knockout cells. Wild-type and Caspase-7 knockout samples were subjected to SDS-PAGE. ab255818 and ab181602 (Rabbit anti-GAPDH loading control) were incubated 1 hour at room temperature at 1/1000 dilution and 1/200,000 dilution respectively. Blots were developed with Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) secondary antibody at 1/100,000 dilution for 1 hour at room temperature before imaging. The blot was developed on a BIO-RAD^® ChemiDoc™ instrument using the ECL technique. Blocking and Diluting Buffer and concentration: 5% NFDM/TBST

Flow cytometric analysis of 4% paraformaldehyde fixed, 90% methanol permeabilized C2C12 (mouse muscle) cells labeling Caspase-7 with ab255818 at 1/50 (Red) compared with a Rabbit monoclonal IgG (ab172730) / Black isotype control and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

Flow cytometric analysis of 4% paraformaldehyde fixed, 90% methanol permeabilized HeLa (human cervix adenocarcinoma) cells labeling Caspase-7 with ab255818 at 1/50 (Red) compared with a Rabbit monoclonal IgG (ab172730) / Black isotype control and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

Immunohistochemical analysis of paraffin-embedded rat colon tissue labeling Caspase-7 with ab255818 at 1/2000 dilution (0.28 ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on rat colon (PMID: 30448733) is observed. The section was incubated with ab255818 for 15 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with hematoxylin. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Immunohistochemical analysis of paraffin-embedded mouse colon tissue labeling Caspase-7 with ab255818 at 1/2000 dilution (0.28 ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on mouse colon (PMID: 23572575) is observed. The section was incubated with ab255818 for 15 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with hematoxylin. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Immunohistochemical analysis of paraffin-embedded human colon tissue labeling Caspase-7 with ab255818 at 1/2000 dilution (0.28 ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on human colon (PMID: 30448733) is observed. The section was incubated with ab255818 for 15 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with hematoxylin. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

Secondary antibody only control/ Used PBS instead of primary antibody, secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).