Anti-CBL antibody [YE323] - C-terminal

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-CBL antibody [YE323] - C-terminal (AB32027)

Intracellular Flow Cytometry analysis of Jurkat (Human T cell leukemia T lymphocyte) cells labeling CBL with purified ab32027 at 1/30 dilution (10 μg/mL) (Red). Cells were fixed with 4% Paraformaldehyde and permeabilised with 90% Methanol. A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) secondary antibody was used at 1/2000. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).

• WB

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Western blot - Anti-CBL antibody [YE323] - C-terminal (AB32027)

All lanes:

Western blot - Anti-CBL antibody [YE323] - C-terminal (ab32027) at 1/1000 dilution

Lane 1:

Raji (Human Burkitt's lymphoma B lymphocyte) whole cell lysate at 15 µg

Lane 2:

Rat testis lysate at 20 µg

Lane 3:

Rat thymus 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 1/2000 dilution

Predicted band size: 100 kDa

Observed band size: 110 kDa

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• WB

Lab

Western blot - Anti-CBL antibody [YE323] - C-terminal (AB32027)

Lane 1 : Wild-type HAP1 whole cell lysate (20 μg)
Lane 2 : CBL knockout HAP1 whole cell lysate (20 μg)
Lane 3 : THP1 whole cell lysate (20 μg)
Lane 4 : Raji whole cell lysate (20 μg)

Lanes 1 - 4 : Merged signal (red and green). Green - ab32027 observed at 100 kDa. Red - loading control, ab9484, observed at 37 kDa.

Unpurified ab32027 was shown to specifically react with CBL in wild-type HAP1 cells as signal was lost in CBL knockout cells. Wild-type and CBL knockout samples were subjected to SDS-PAGE. ab32027 and ab9484 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1/5000 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-CBL antibody [YE323] - C-terminal (ab32027)

Predicted band size: 100 kDa

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• WB

Lab

Western blot - Anti-CBL antibody [YE323] - C-terminal (AB32027)

Lanes 1-4 : Merged signal (red and green). Green - ab32027 observed at 110 kDa. Red - loading control ab8245 observed at 36 kDa.

Unpurified ab32027 Anti-CBL antibody [YE323] - C-terminal was shown to specifically react with CBL in wild-type HEK293T cells. Loss of signal was observed when knockout cell line ab267245 (knockout cell lysate ab257200) was used. Wild-type and CBL knockout samples were subjected to SDS-PAGE. ab32027 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-CBL antibody [YE323] - C-terminal (ab32027) at 1/1000 dilution

Lane 1:

Wild-type HEK293T cell lysate at 20 µg

Lane 2:

CBL knockout HEK293T cell lysate at 20 µg

Lane 2:

Western blot - Human CBL knockout HEK-293T cell line (<a href='/en-us/products/cell-lines/human-cbl-knockout-hek-293t-cell-line-ab267245'>ab267245</a>)

Lane 3:

THP-1 cell lysate at 20 µg

Lane 4:

Raji cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/10000 dilution

Predicted band size: 100 kDa

Observed band size: 110 kDa

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• WB

Lab

Western blot - Anti-CBL antibody [YE323] - C-terminal (AB32027)

Western blot : Anti-CBL antibody [YE323] (ab32027) staining at 1/1000 dilution, shown in green; Mouse anti-CANX [CANX/1543] (ab238078) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab32027 was shown to bind specifically to CBL. A band was observed at 105 kDa in wild-type HCT 116 cell lysates with no signal observed at this size in CBL knockout cell line. To generate this image, wild-type and CBL knockout HCT 116 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % 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 IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-CBL antibody [YE323] - C-terminal (ab32027) at 1/1000 dilution

Lane 1:

Wild-type HCT 116 cell lysate at 20 µg

Lane 2:

CBL knockout HCT 116 cell lysate at 20 µg

Lane 2:

Western blot - Human CBL knockout HCT116 cell line (<a href='/en-us/products/cell-lines/human-cbl-knockout-hct116-cell-line-ab287255'>ab287255</a>)

Secondary

All lanes:

Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

Observed band size: 105 kDa

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• WB

Lab

Western blot - Anti-CBL antibody [YE323] - C-terminal (AB32027)

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

Exposure time : 30 seconds

All lanes:

Western blot - Anti-CBL antibody [YE323] - C-terminal (ab32027) at 1/1000 dilution

Lane 1:

WEHI-231 (Mouse B cell lymphoma B lymphocyte) cell lysate at 10 µg

Lane 2:

F9 (Mouse embryonal carcinoma epithelial cell) cell lysate at 10 µg

Lane 3:

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

Lane 4:

Mouse thymus lysate at 10 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

Predicted band size: 100 kDa

Observed band size: 110 kDa

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