Anti-Caspase-3 antibody [RM1299]

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Caspase-3 antibody [RM1299] (AB323369)

Immunohistochemical analysis of paraffin-embedded Human skeletal muscle tissue labeling Caspase-3 with ab323369 at 1/5000 (0.10 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Low expression : no staining on human skeletal muscle.
The section was incubated with ab323369 for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument

Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Citrate buffer (pH 6.0, Epitope Retrieval Solution 1) for 20 mins

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Caspase-3 antibody [RM1299] (AB323369)

Immunohistochemical analysis of paraffin-embedded Human tonsil tissue labeling Caspase-3 with ab323369 at 1/5000 (0.10 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on human tonsil.
The section was incubated with ab323369 for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument

Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Citrate buffer (pH 6.0, Epitope Retrieval Solution 1) for 20 mins

• IP

Supplier Data

Immunoprecipitation - Anti-Caspase-3 antibody [RM1299] (AB323369)

Caspase-3 was immunoprecipitated from 0.35 mg parental HAP1(HOWT01) (human chronic myelogenous leukemia near-haploid cell ) whole cell lysate with ab323369 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab323369 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

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

All lanes:

Immunoprecipitation - Anti-Caspase-3 antibody [RM1299] (ab323369) at 1/1000 dilution

Lane 1:

parental HAP1(HOWT01) (human chronic myelogenous leukemia near-haploid cell ) whole cell lysate at 10 µg

Lane 2:

ab323369 at 1/30 IP in parental HAP1(HOWT01) (human chronic myelogenous leukemia near-haploid cell ) whole cell lysate

Lane 3:

Rabbit monoclonal IgG (<a href='/en-us/products/primary-antibodies/rabbit-igg-monoclonal-epr25a-isotype-control-ab172730'>ab172730</a>) instead of ab323369 in parental HAP1(HOWT01) whole cell lysate

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

false

Exposure time: 6s

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Caspase-3 antibody [RM1299] (AB323369)

Immunohistochemical analysis of paraffin-embedded Mouse cardiac muscle tissue labeling Caspase-3 with ab323369 at 1/5000 (0.10 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Low expression : nearly no staining on mouse cardiac muscle.
The section was incubated with ab323369 for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument

Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Citrate buffer (pH 6.0, Epitope Retrieval Solution 1) for 20 mins

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Caspase-3 antibody [RM1299] (AB323369)

Immunohistochemical analysis of paraffin-embedded Mouse colon tissue labeling Caspase-3 with ab323369 at 1/5000 (0.10 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on mouse colon.
The section was incubated with ab323369 for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument

Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Citrate buffer (pH 6.0, Epitope Retrieval Solution 1) for 20 mins

• IP

Supplier Data

Immunoprecipitation - Anti-Caspase-3 antibody [RM1299] (AB323369)

Caspase-3 was immunoprecipitated from 0.35 mg NIH/3T3 (mouse embryonic fibroblast) whole cell lysate with ab323369 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab323369 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

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

All lanes:

Immunoprecipitation - Anti-Caspase-3 antibody [RM1299] (ab323369) at 1/1000 dilution

Lane 1:

NIH/3T3 (mouse embryonic fibroblast) whole cell lysate at 10 µg

Lane 2:

ab323369 at 1/30 IP in NIH/3T3 (mouse embryonic fibroblast) whole cell lysate

Lane 3:

Rabbit monoclonal IgG (<a href='/en-us/products/primary-antibodies/rabbit-igg-monoclonal-epr25a-isotype-control-ab172730'>ab172730</a>) instead of ab323369 in NIH/3T3 whole cell lysate

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

false

Exposure time: 32s

• WB

Supplier Data

Western blot - Anti-Caspase-3 antibody [RM1299] (AB323369)

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

The molecular weight observed is consistent with what has been described in the literature (PMID : 11423986).

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

Exposure time : Lane 1-2 : 70 seconds; Lane 3-4 : 15 seconds

All lanes:

Western blot - Anti-Caspase-3 antibody [RM1299] (ab323369) at 1/1000 dilution

Lane 1:

Untreated HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

HeLa treated with 2uM staurosporine for 3 hours, whole cell lysate at 20 µg

Lane 3:

Untreated Jurkat (human T cell leukemia T lymphocyte from peripheral blood) whole cell lysate at 20 µg

Lane 4:

Jurkat treated with 1uM staurosporine for 3 hours, whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Observed band size: 32 kDa,17 kDa,36 kDa

false

• WB

Supplier Data

Western blot - Anti-Caspase-3 antibody [RM1299] (AB323369)

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

In Western blot, ab323369 was shown to bind specifically to Caspase-3. Target of interest was observed at 32 kDa in wild-type HAP1 cell lysates (lane 1) with no signal observed at this size in Caspase-3 knockout cell line (lane 2).

In Western blot, Anti-Histone H3 antibody [EPR16987] - Nuclear Marker and ChIP Grade (ab176842) staining at 1/100000 dilution.

All lanes:

Western blot - Anti-Caspase-3 antibody [RM1299] (ab323369) at 1/1000 dilution

Lane 1:

Parental HAP1(HOWT01) (human chronic myelogenous leukemia near-haploid cell) whole cell lysate at 20 µg

Lane 2:

Caspase-3 knockout HAP1 whole cell lysate at 20 µg

Lane 3:

HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 4:

Jurkat (human T cell leukemia T lymphocyte from peripheral blood) whole cell lysate at 20 µg

Lane 5:

293T (human embryonic kidney epithelial cell) whole cell lysate at 20 µg

Lane 6:

Neuro-2a (mouse neuroblastoma neuroblast) whole cell lysate at 20 µg

Lane 7:

NIH/3T3 (mouse embryonic fibroblast) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Observed band size: 32 kDa,15 kDa

false

Exposure time: 26s

• WB

Supplier Data

Western blot - Anti-Caspase-3 antibody [RM1299] (AB323369)

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

Low expression : skeletal muscle

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

All lanes:

Western blot - Anti-Caspase-3 antibody [RM1299] (ab323369) at 1/1000 dilution

Lane 1:

Human tonsil tissue lysate at 20 µg

Lane 2:

Human skeletal muscle tissue lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Observed band size: 32 kDa,36 kDa

false

Exposure time: 15s

• WB

Supplier Data

Western blot - Anti-Caspase-3 antibody [RM1299] (AB323369)

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

Low expression : heart

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

All lanes:

Western blot - Anti-Caspase-3 antibody [RM1299] (ab323369) at 1/1000 dilution

Lane 1:

Human stomach tissue lysate at 20 µg

Lane 2:

Human spleen tissue lysate at 20 µg

Lane 3:

Human testis tissue lysate at 20 µg

Lane 4:

Mouse lung tissue at 20 µg

Lane 5:

Mouse spleen tissue lysate at 20 µg

Lane 6:

Mouse heart tissue lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Observed band size: 32 kDa,36 kDa

false

Exposure time: 37s

• WB

Supplier Data

Western blot - Anti-Caspase-3 antibody [RM1299] (AB323369)

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

The molecular weight observed is consistent with what has been described in the literature (PMID : 11423986).

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

All lanes:

Western blot - Anti-Caspase-3 antibody [RM1299] (ab323369) at 1/1000 dilution

Lane 1:

Untreated NIH/3T3 (mouse embryonic fibroblast) whole cell lysate at 20 µg

Lane 2:

NIH/3T3 treated with 2uM staurosporine for 6 hours, whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Observed band size: 32 kDa,17 kDa,36 kDa

false

Exposure time: 6s