Anti-A-Raf antibody [EPR25405-17]

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-A-Raf antibody [EPR25405-17] (AB314539)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized MEF (mouse embryo fibroblast) cells labelling A-Raf with ab314539 at 1/100 (5.35 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1006 (2ug/ml) dilution (Green). Confocal image showing mitochondrial staining in MEF cells. 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 (Red). 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 1/1006 (2ug/ml) dilution.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-A-Raf antibody [EPR25405-17] (AB314539)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized NIH/3T3 (mouse embryonic fibroblast) cells labelling A-Raf with ab314539 at 1/100 (5.35 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1007 (2ug/ml) dilution (Green). Confocal image showing mitochondrial staining in NIH/3T3 cells. 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 (Red). 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 1/1007 (2ug/ml) dilution.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-A-Raf antibody [EPR25405-17] (AB314539)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized NIH/3T3 (mouse embryonic fibroblast) cells labelling A-Raf with ab314539 at 1/50 dilution (1 ug, 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). A Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/5000 dilution was used as the secondary antibody.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-A-Raf antibody [EPR25405-17] (AB314539)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized MEF (mouse embryo fibroblast) cells labelling A-Raf with ab314539 at 1/50 dilution (1 ug, 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). A Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/5000 dilution was used as the secondary antibody.

• WB

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Western blot - Anti-A-Raf antibody [EPR25405-17] (AB314539)

Blocking and diluting buffer and concentration : 5% NFDM/TBST Lysates were freshly made and used for Western blotting immediately to minimize protein degradation.

All lanes:

Western blot - Anti-A-Raf antibody [EPR25405-17] (ab314539) at 1/1000 dilution

Lane 1:

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

Lane 2:

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

Lane 3:

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

Lane 4:

PC-12 (rat adrenal gland pheochromocytoma cell) 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 1/20000 dilution

Observed band size: 68 kDa

false

Exposure time: 92s

• WB

Supplier Data

Western blot - Anti-A-Raf antibody [EPR25405-17] (AB314539)

Blocking and diluting buffer and concentration : 5% NFDM/TBST The identity of the lower MW band at approximately 42 kDa (in lane 3) is unknown. In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

All lanes:

Western blot - Anti-A-Raf antibody [EPR25405-17] (ab314539) at 1/1000 dilution

Lane 1:

NIH/3T3 (mouse embryonic fibroblast) transfected with scrambled siRNA control whole cell lysate at 20 µg

Lane 2:

NIH/3T3 transfected with siRNA specifically targeti A-Raf whole cell lysate at 20 µg

Lane 3:

MEF (mouse embryo 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 1/20000 dilution

Observed band size: 68 kDa

false

Exposure time: 180s

• WB

Supplier Data

Western blot - Anti-A-Raf antibody [EPR25405-17] (AB314539)

Blocking and diluting buffer and concentration : 5% NFDM/TBST The identity of the bands between 10 kDa and 68 kDa are unknown. Exposure time : Lane 1 : 26 seconds; Lanes 2-3 : 180 seconds.

All lanes:

Western blot - Anti-A-Raf antibody [EPR25405-17] (ab314539) at 1/1000 dilution

Lane 1:

Mouse epididymis tissue lysate at 20 µg

Lane 2:

Mouse prostate tissue lysate at 20 µg

Lane 3:

C2C12 (mouse myoblast) 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 1/20000 dilution

Observed band size: 68 kDa

false

• WB

Supplier Data

Western blot - Anti-A-Raf antibody [EPR25405-17] (AB314539)

Blocking and diluting buffer and concentration : 5% NFDM/TBST This antibody does not cross-react with mouse B-Raf and c-Raf. In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution. In Western blot, Anti-6X His tag® antibody [EPR20547] - ChIP Grade (ab213204) staining at 1/5000 dilution.

All lanes:

Western blot - Anti-A-Raf antibody [EPR25405-17] (ab314539) at 1/1000 dilution

Lane 1:

293T (human embryonic kidney epithelial cell) cells transfected with an empty vector containing a His-tag, whole cell lysate at 5 µg

Lane 2:

293T cells transfected with a mouse A-Raf expression vector containing a His-tag, whole cell lysate at 5 µg

Lane 3:

293T cells transfected with a mouse c-Raf expression vector containing a His-tag, whole cell lysate at 5 µg

Lane 4:

293T cells transfected with a mouse B-Raf expression vector containing a His-tag, whole cell lysate at 5 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: 68 kDa

false

Exposure time: 3s