Anti-Bak antibody [EPR27487-15]

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Bak antibody [EPR27487-15] (AB317254)

Immunohistochemical analysis of paraffin-embedded Mouse colon tissue labeling Bak with ab317254 at 1/500 (1.004 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining in mouse colon.
The section was incubated with ab317254 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 Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Bak antibody [EPR27487-15] (AB317254)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized C2C12 (mouse myoblast) cells labelling Bak with ab317254 at 1/500 (1.004 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 (2 ug/ml) dilution (Green).

Confocal image showing mitochondrial staining in C2C12 cell line (shown in green). The counterstain was observed in magenta. Nuclear DNA was labeled with DAPI (shown in blue).
Image was taken with a confocal microscope(Leica-Microsystems, TCS SP8).

anti-COX IV mouse monoclonal antibody - Mitochondrial Marker was used to counterstain at 1/200 (2.5ug/ml) dilution (Magenta). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 (2 ug/ml) dilution.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Bak antibody [EPR27487-15] (AB317254)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized C2C12 (mouse myoblast) cells labelling Bak with ab317254 at 1/500 dilution (0.1ug)/Red compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue).

Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/5000 dilution was used as the secondary antibody.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Bak antibody [EPR27487-15] (AB317254)

Immunohistochemical analysis of paraffin-embedded Mouse breast cancer tissue labeling Bak with ab317254 at 1/500 (1.004 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining in mouse breast cancer.
The section was incubated with ab317254 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 Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Bak antibody [EPR27487-15] (AB317254)

Immunohistochemical analysis of paraffin-embedded Mouse spleen tissue labeling Bak with ab317254 at 1/500 (1.004 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining in mouse spleen.
The section was incubated with ab317254 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 Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

• IP

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Immunoprecipitation - Anti-Bak antibody [EPR27487-15] (AB317254)

Bak was immunoprecipitated from 0.35 mg C2C12 (mouse myoblast) whole cell lysate with ab317254 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab317254 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : C2C12 (mouse myoblast) whole cell lysate
Lane 2 : ab317254 IP in C2C12 (mouse myoblast) whole cell lysate
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab317254 in C2C12 whole cell lysate.

All lanes:

Immunoprecipitation - Anti-Bak antibody [EPR27487-15] (ab317254) at 1/30 dilution

All lanes:

C2C12 (mouse myoblast) 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: 8s

• WB

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Western blot - Anti-Bak antibody [EPR27487-15] (AB317254)

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

Exposure time : Lanes 1-4 : 15 seconds, lane 5 : 48 seconds

All lanes:

Western blot - Anti-Bak antibody [EPR27487-15] (ab317254) at 1/1000 dilution

Lane 1:

C2C12 (mouse myoblast) transfected with scrambled siRNA control whole cell lysate at 20 µg

Lane 2:

C2C12 transfected with siRNA specifically targeti Bak whole cell lysate at 20 µg

Lane 3:

A20 (mouse reticum sarcoma b lymphocyte) whole cell lysate at 20 µg

Lane 4:

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

Lane 5:

Mouse spleen 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: 23 kDa,36 kDa

false