All lanes : Anti-Caspase-6/CASP-6 antibody [EPR18043] (ab185645) at 1/1000 dilution

Lane 1 : Untreated Jurkat (Human T cell leukemia cells from peripheral blood) whole cell lysate
Lane 2 : Jurkat (Human T cell leukemia cells from peripheral blood) whole cell lysate 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: 33 kDa
Observed band size: 11,33 kDa why is the actual band size different from the predicted?


Exposure time: 1 minute

Blocking/Dilution buffer: 5% NFDM/TBST.

All lanes : Anti-Caspase-6/CASP-6 antibody [EPR18043] (ab185645) at 1/1000 dilution

Lane 1 : Untreated NIH/3T3 (Mouse embyro fibroblast cells) whole cell lysate
Lane 2 : NIH/3T3 (Mouse embyro fibroblast cells) whole cell lysate 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: 33 kDa
Observed band size: 11,33 kDa why is the actual band size different from the predicted?


Exposure time: 1 minute

The predicted MW is 32kDa for mouse and rat full-length procaspase-6.

Blocking/Dilution buffer: 5% NFDM/TBST.

All lanes : Anti-Caspase-6/CASP-6 antibody [EPR18043] (ab185645) at 1/1000 dilution

Lane 1 : C6 (Rat glial tumor cells) whole cell lysate
Lane 2 : PC-12 (Rat adrenal gland pheochromocytoma) whole cell lysate

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: 33 kDa
Observed band size: 33 kDa


Exposure time: 1 minute

The predicted MW is 32kDa for mouse and rat full-length procaspase-6.

Blocking/Dilution buffer: 5% NFDM/TBST.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized Jurkat (Human T cell leukemia cells from peripheral blood) cells labeling Caspase-6/CASP-6 with ab185645 at 1/500 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/500 dilution (green).

Confocal image showing both nuclear and cytoplasmic staining on Jurkat cells. The staining remained similar after treatment with staurosporine (1uM, 4 hours) as the antibody interacts with the subunit p11. 

The nuclear counter stain is DAPI (blue).

Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution and ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution (red).
The negative controls are as follows:
-ve control 1: ab185645 at 1/500 dilution followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution.
-ve control 2: ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution followed by ab150077 (Alexa Fluor®488 Goat Anti-Rabbit IgG H&L) at 1/500 dilution.

Immunohistochemical analysis of paraffin-embedded Human colon tissue labeling Caspase-6/CASP-6 with ab185645 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody at 1/500 dilution.

Cytoplasmic and nuclear staining on epithelial cells of human colon 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 Mouse brain tissue labeling Caspase-6/CASP-6 with ab185645 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody at 1/500 dilution.

Cytoplasmic staining on neurons of mouse brain 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 Rat brain tissue labeling Caspase-6/CASP-6 with ab185645 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody at 1/500 dilution.

Cytoplasmic staining on neurons of rat brain 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.

Caspase-6/CASP-6 was immunoprecipitated from 1mg of NIH/3T3 (Mouse embyro fibroblast cells) treated with 1uM staurosporine for 4 hours whole cell lysate with ab185645 at 1/50 dilution. Western blot was performed from the immunoprecipitate using ab185645 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used for detection at 1/1500 dilution.

Lane 1: NIH/3T3 treated with 1uM staurosporine for 4 hours whole cell lysate10 µg (Input). Lane 2: ab185645 IP in NIH/3T3 treated with 1uM staurosporine for 4 hours whole cell lysate. Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab185645 in NIH/3T3 treated with 1uM staurosporine for 4 hours whole cell lysate.

The cleaved Caspase-6/CASP-6 appears slightly larger than 11kDa. This fragment contains subunit p11 plus the internal propeptide.

Blocking and dilution buffer and concentration: 5% NFDM/TBST.