Anti-Caspase-6/CASP-6 抗体 [EPR18043]

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Caspase-6/CASP-6 antibody [EPR18043] (AB185645)

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.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Caspase-6/CASP-6 antibody [EPR18043] (AB185645)

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.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Caspase-6/CASP-6 antibody [EPR18043] (AB185645)

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.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Caspase-6/CASP-6 antibody [EPR18043] (AB185645)

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.

• IP

Supplier Data

Immunoprecipitation - Anti-Caspase-6/CASP-6 antibody [EPR18043] (AB185645)

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.

All lanes:

Immunoprecipitation - Anti-Caspase-6/CASP-6 antibody [EPR18043] (ab185645)

Predicted band size: 33 kDa

Observed band size: 11 kDa,33 kDa

false

• WB

Supplier Data

Western blot - Anti-Caspase-6/CASP-6 antibody [EPR18043] (AB185645)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - 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 at 10 µg

Lane 2:

Jurkat (Human T cell leukemia cells from peripheral blood) whole cell lysate treated with 1uM staurosporine for 4 hours at 10 µg

Secondary

All lanes:

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

Predicted band size: 33 kDa

Observed band size: 11 kDa,33 kDa

false

Exposure time: 1min

• WB

Lab

Western blot - Anti-Caspase-6/CASP-6 antibody [EPR18043] (AB185645)

Western blot : Anti-Caspase-6/CASP-6 antibody [EPR18043] ab185645 staining at 1/1000 dilution, shown in green; Mouse anti alpha Tubulin ab7291 loading control staining at 1/20000 dilution, shown in magenta. A band was observed at 33 kDa in Wild-type HAP1 cell lysates with no signal observed at this size in CASP6 knockout HAP1 cell line. To generate this image, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 5% Milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit 800CW and Goat anti-Mouse 680RD at 1/20,000 dilution.

All lanes:

Western blot - Anti-Caspase-6/CASP-6 antibody [EPR18043] (ab185645) at 1/1000 dilution

Lane 1:

Wild-type HAP1 cell lysates at 20 µg

Lane 2:

CASP6 knockout HAP1 cell lysates at 20 µg

Lane 3:

MCF7 cell lysates at 20 µg

Lane 4:

Jurkat cell lysates at 20 µg

Secondary

All lanes:

Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20000 dilution

Predicted band size: 33 kDa

Observed band size: 33 kDa

false

• WB

Supplier Data

Western blot - Anti-Caspase-6/CASP-6 antibody [EPR18043] (AB185645)

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

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-Caspase-6/CASP-6 antibody [EPR18043] (ab185645) at 1/1000 dilution

Lane 1:

C6 (Rat glial tumor cells) whole cell lysate at 10 µg

Lane 2:

PC-12 (Rat adrenal gland pheochromocytoma) whole cell lysate at 10 µg

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

false

Exposure time: 1min

• WB

Supplier Data

Western blot - Anti-Caspase-6/CASP-6 antibody [EPR18043] (AB185645)

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

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-Caspase-6/CASP-6 antibody [EPR18043] (ab185645) at 1/1000 dilution

Lane 1:

Untreated NIH/3T3 (Mouse embyro fibroblast cells) whole cell lysate at 10 µg

Lane 2:

NIH/3T3 (Mouse embyro fibroblast cells) whole cell lysate treated with 1uM staurosporine for 4 hours at 10 µg

Secondary

All lanes:

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

Predicted band size: 33 kDa

Observed band size: 11 kDa,33 kDa

false

Exposure time: 1min