Anti-CBL antibody [17/c-Cbl] - BSA and Azide free

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CBL antibody [17/c-Cbl] - BSA and Azide free (AB309550)

This data was developed using ab309549 the same antibody clone in a different buffer formulation. Immunohistochemical analysis of paraffin-embedded Human testis tissue labeling CBL with ab309549 at 1/5000 (0.1968 ug/ml) dilution followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Cytoplasmic staining on human testis.The section was incubated with ab309549 for 30 mins at room temperature, followed by anti-mouse IgG1 antibody (ab125913) for 8 mins during the LeicaDS9800 kit staining procedure.The immunostaining was performed on a Leica Biosystems BOND® RX instrument Counterstained with Hematoxylin. 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 [17/c-Cbl] - BSA and Azide free (AB309550)

This data was developed using ab309549 the same antibody clone in a different buffer formulation. Immunohistochemical analysis of paraffin-embedded Human tonsil tissue labeling CBL with ab309549 at 1/5000 (0.1968 ug/ml) dilution followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Cytoplasmic staining on human tonsil.The section was incubated with ab309549 for 30 mins at room temperature, followed by anti-mouse IgG1 antibody (ab125913) for 8 mins during the LeicaDS9800 kit staining procedure.The immunostaining was performed on a Leica Biosystems BOND® RX instrument Counterstained with Hematoxylin. 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 [17/c-Cbl] - BSA and Azide free (AB309550)

This data was developed using ab309549 the same antibody clone in a different buffer formulation. Immunohistochemical analysis of paraffin-embedded Rat spleen tissue labeling CBL with ab309549 at 1/5000 (0.1968 ug/ml) dilution followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Cytoplasmic staining on rat spleen.The section was incubated with ab309549 for 30 mins at room temperature, followed by anti-mouse IgG1 antibody (ab125913) for 8 mins during the LeicaDS9800 kit staining procedure.The immunostaining was performed on a Leica Biosystems BOND® RX instrument Counterstained with Hematoxylin. 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 [17/c-Cbl] - BSA and Azide free (AB309550)

This data was developed using ab309549 the same antibody clone in a different buffer formulation. Immunohistochemical analysis of paraffin-embedded Mouse spleen tissue labeling CBL with ab309549 at 1/5000 (0.1968 ug/ml) dilution followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Cytoplasmic staining on mouse spleen.The section was incubated with ab309549 for 30 mins at room temperature, followed by anti-mouse IgG1 antibody (ab125913) for 8 mins during the LeicaDS9800 kit staining procedure.The immunostaining was performed on a Leica Biosystems BOND® RX instrument Counterstained with Hematoxylin. 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 [17/c-Cbl] - BSA and Azide free (AB309550)

This data was developed using ab309549, the same antibody clone in a different buffer formulation.Blocking and diluting buffer and concentration : 5% NFDM/TBST Low expression : NIH/3T3 (PMID : 12193575). Performed under reducing conditions. Frozen lysates were used for Western blotting. In Western blot, ab309549 was shown to bind specifically to CBL. Target of interest was observed at 120 kDa wild-type 293T cell lysates (lane 1) with no signal observed at this size in CBL knockout cell line (lane 2, knockout cell line ab267245 / knockout cell lysate ab257200). The band beneath the target band (120 kDa) is likely to be degraded target fragment. In Western blot, anti-GAPDH antibody (ab181602) loading control staining at 1/200000 dilution. Exposure time : 3 minutes

All lanes:

Western blot - Anti-CBL antibody [17/c-Cbl] (<a href='/en-us/products/primary-antibodies/cbl-antibody-17-c-cbl-ab309549'>ab309549</a>) at 1/1000 dilution

Lane 1:

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

Lane 3:

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

Lane 4:

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

Lane 5:

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

Secondary

All lanes:

Peroxidase-Conjugated Goat anti-Mouse IgG (H+L) (ZB-2305) at 1/10000 dilution

Observed band size: 120 kDa

false

Exposure time: 3min

• WB

Supplier Data

Western blot - Anti-CBL antibody [17/c-Cbl] - BSA and Azide free (AB309550)

This data was developed using ab309549, the same antibody clone in a different buffer formulation. Blocking and diluting buffer and concentration : 5% NFDM/TBST The lysates were freshly made and used for Western blotting immediately to minimize protein degradation. Exposure time : 48 seconds

All lanes:

Western blot - Anti-CBL antibody [17/c-Cbl] (<a href='/en-us/products/primary-antibodies/cbl-antibody-17-c-cbl-ab309549'>ab309549</a>) at 1/1000 dilution

Lane 1:

Raji (human Burkitts lymphoma B lymphocyte) whole cell lysate at 20 µg

Lane 2:

Mouse testis tissue lysate at 20 µg

Secondary

All lanes:

Peroxidase-Conjugated Goat anti-Mouse IgG (H+L) (ZB-2305) at 1/10000 dilution

Observed band size: 120 kDa

false

Exposure time: 48s

• WB

Supplier Data

Western blot - Anti-CBL antibody [17/c-Cbl] - BSA and Azide free (AB309550)

This data was developed using ab309549, the same antibody clone in a different buffer formulation.Blocking and diluting buffer and concentration : 5% NFDM/TBST This target was tested in fresh and frozen Ramos, and it was concluded that CBL is the easy target for degradation. Lane2 was freshly made and used for Western blotting immediately to minimize protein degradation. Exposure time : 48 seconds

All lanes:

Western blot - Anti-CBL antibody [17/c-Cbl] (<a href='/en-us/products/primary-antibodies/cbl-antibody-17-c-cbl-ab309549'>ab309549</a>) at 1/1000 dilution

All lanes:

Ramos (human Burkitts lymphoma B lymphocyte) whole cell lysate at 20 µg

Secondary

All lanes:

Peroxidase-Conjugated Goat anti-Mouse IgG (H+L) (ZB-2305) at 1/10000 dilution

Observed band size: 120 kDa

false

Exposure time: 48s

• WB

Lab

Western blot - Anti-CBL antibody [17/c-Cbl] - BSA and Azide free (AB309550)

This data was developed using the same antibody clone in a different buffer formulation (ab309549).

Western blot : Anti-CBL antibody [17/c-Cbl] (ab309549) staining at 1/1000 dilution, shown in green; Rabbit anti-alpha Tubulin antibody [EP1332Y] (ab52866) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab309549 was shown to bind specifically to CBL. A band was observed at 105-110 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 5 % 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-Mouse IgG H&L 800CW and Goat anti-Rabbit IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-CBL antibody [17/c-Cbl] (<a href='/en-us/products/primary-antibodies/cbl-antibody-17-c-cbl-ab309549'>ab309549</a>) 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

Secondary

All lanes:

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

Observed band size: 105 kDa,110 kDa

false

• WB

Supplier Data

Western blot - Anti-CBL antibody [17/c-Cbl] - BSA and Azide free (AB309550)

This data was developed using ab309549, the same antibody clone in a different buffer formulation. Blocking and diluting buffer and concentration : 5% NFDM/TBST Exposure time : 40 seconds

All lanes:

Western blot - Anti-CBL antibody [17/c-Cbl] (<a href='/en-us/products/primary-antibodies/cbl-antibody-17-c-cbl-ab309549'>ab309549</a>) at 1/1000 dilution

Lane 1:

Rat lymph node tissue lysate at 20 µg

Lane 2:

Rat testis tissue lysate at 20 µg

Lane 3:

WEHI-3 (mouse leukemia lymphoblast) whole cell lysate at 20 µg

Secondary

All lanes:

Peroxidase-Conjugated Goat anti-Mouse IgG (H+L) (ZB-2305) at 1/10000 dilution

Observed band size: 120 kDa

false

Exposure time: 40s