Anti-CBL antibody [EPR25388-126]

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CBL antibody [EPR25388-126] (AB324458)

Immunohistochemical analysis of paraffin-embedded Human skeletal muscle tissue labeling CBL with ab324458 at 1/100 (4.71 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Low expression tissue : no staining on human skeletal muscle. The primary antibody was incubated 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-CBL antibody [EPR25388-126] (AB324458)

Immunohistochemical analysis of paraffin-embedded (A) Parental 293T (human embryonic kidney epithelial cell) cell pellet and (B) CBL knockout 293T cell pellet labeling CBL with ab324458 at 1/2000 (0.236 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on (A) Parental 293T cell pellet, no staining on (B) CBL knockout 293T cell pellet. The primary antibody was incubated for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Slides were incubated with 3% Hydrogen Peroxide for 10 mins at room temperature after secondary antibody incubation to reduce the background

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-CBL antibody [EPR25388-126] (AB324458)

Immunohistochemical analysis of paraffin-embedded Human testis tissue labeling CBL with ab324458 at 1/100 (4.71 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on human testis. The primary antibody was incubated 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-CBL antibody [EPR25388-126] (AB324458)

Immunohistochemical analysis of paraffin-embedded Human tonsil tissue labeling CBL with ab324458 at 1/100 (4.71 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on human tonsil. The primary antibody was incubated 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-CBL antibody [EPR25388-126] (AB324458)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized CBL KO HEK293T (CBL knockout human embryonic kidney epithelial cell), ab267245 cells labelling CBL with ab324458 at 1/100 (4.71 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 cytoplasmic staining in wildtype HEK-293T, and showing no staining in CBL knockout HEK-293T cells (shown in green). The counterstain was observed in magenta. Nuclear DNA was labelled with DAPI (shown in blue).
Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

ab7291 Anti-alpha Tubulin mouse monoclonal antibody was used to counterstain tubulin at 1/1000 (1 ug/ml) dilution, followed by ab150120 Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) at 1/1000 (2 ug/ml) dilution (Magenta).

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-CBL antibody [EPR25388-126] (AB324458)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized EL4 (mouse lymphoma T lymphocyte) cells labelling CBL with ab324458 at 1/100 (4.71 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 cytoplasmic staining in EL4 cell line (shown in green). The counterstain was observed in magenta. Nuclear DNA was labelled with DAPI (shown in blue).
Low expression : NIH/3T3.
Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

ab7291 Anti-alpha Tubulin mouse monoclonal antibody was used to counterstain tubulin at 1/1000 (1 ug/ml) dilution, followed by ab150120 Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) at 1/1000 (2 ug/ml) dilution (Magenta).

-ve control 1 : ab324458 at a 1/100 dilution followed by ab150120 at a 1/1000 dilution.

-ve control 2 : ab7291 at a 1/1000 dilution followed by ab150081 at a 1/1000 dilution.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CBL antibody [EPR25388-126] (AB324458)

Immunohistochemical analysis of paraffin-embedded Mouse testis tissue labeling CBL with ab324458 at 1/2000 (0.236 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on mouse testis. The primary antibody was incubated for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Slides were incubated with 3% Hydrogen Peroxide for 10 mins at room temperature after secondary antibody incubation to reduce the background

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-CBL antibody [EPR25388-126] (AB324458)

Immunohistochemical analysis of paraffin-embedded Mouse skeletal muscle tissue labeling CBL with ab324458 at 1/2000 (0.236 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Low expression tissue : no staining on mouse skeletal muscle. The primary antibody was incubated for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument.Slides were incubated with 3% Hydrogen Peroxide for 10 mins at room temperature after secondary antibody incubation to reduce the background

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-CBL antibody [EPR25388-126] (AB324458)

Immunohistochemical analysis of paraffin-embedded Mouse spleen tissue labeling CBL with ab324458 at 1/2000 (0.236 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on mouse spleen. The primary antibody was incubated for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Slides were incubated with 3% Hydrogen Peroxide for 10 mins at room temperature after secondary antibody incubation to reduce the background

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-CBL antibody [EPR25388-126] (AB324458)

Immunohistochemical analysis of paraffin-embedded Mouse large B cell lymphoma tissue labeling CBL with ab324458 at 1/2000 (0.236 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on mouse large B cell lymphoma. The primary antibody was incubated for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Slides were incubated with 3% Hydrogen Peroxide for 10 mins at room temperature after secondary antibody incubation to reduce the background

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

• WB

Supplier Data

Western blot - Anti-CBL antibody [EPR25388-126] (AB324458)

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

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

The bands beneath the target band (110 kDa) are likely to be truncated froms or degraded target fragments.

All lanes:

Western blot - Anti-CBL antibody [EPR25388-126] (ab324458) at 1/1000 dilution

All lanes:

Human testis tissue lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

Observed band size: 110 kDa,36 kDa

false

Exposure time: 180s

• WB

Supplier Data

Western blot - Anti-CBL antibody [EPR25388-126] (AB324458)

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

Performed under reducing conditions.

In Western blot, ab324458 was shown to bind specifically to CBL. Target of interest was observed at 110 kDa (if only a single band) in wild-type HEK-293T cell lysates (lane 1) with no signal observed at this size in CBL knockout cell line (lane 2) (lane 2, knockout cell line ab267245 / knockout cell lysate ab257200).

The bands beneath the target band (110 kDa) are likely to be truncated froms or degraded target fragments.

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

All lanes:

Western blot - Anti-CBL antibody [EPR25388-126] (ab324458) at 1/1000 dilution

Lane 1:

Wild-type HEK-293T (human embryonic kidney epithelial cell) whole cell lysate at 20 µg

Lane 2:

CBL knockout HEK-293T whole cell lysate at 20 µg

Lane 3:

HCT 116 (human colorectal carcinoma epithelial cell) whole cell lysate at 20 µg

Lane 4:

Raji (human burkitt's lymphoma b lymphocyte) 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: 110 kDa,36 kDa

false

Exposure time: 15s

• WB

Supplier Data

Western blot - Anti-CBL antibody [EPR25388-126] (AB324458)

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

Low expression : NIH/3T3, mouse skeletal muscle.

The bands beneath the target band (110 kDa) are likely to be truncated froms or degraded target fragments.

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

Exposure time : Lane 1 : 15 seconds; Lanes 2-3 : 3 seconds; Lanes 4-6 : 8 seconds.

All lanes:

Western blot - Anti-CBL antibody [EPR25388-126] (ab324458) at 1/1000 dilution

Lane 1:

F9 (mouse embryonal carcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

EL4 (mouse lymphoma t lymphocyte) whole cell lysate at 20 µg

Lane 3:

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

Lane 4:

Mouse thymus tissue lysate at 20 µg

Lane 5:

Mouse skeletal muscle tissue lysate at 20 µg

Lane 6:

Mouse testis 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 1/20000 dilution

Observed band size: 110 kDa,36 kDa

false