Anti-CSK 抗体 [EPR24673-97] (ab300132)

Western blot: Anti-CSK antibody [EPR24673-97] (ab300132) staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab300132 was shown to bind specifically to CSK. A band was observed at 50 kDa in wild-type A549 cell lysates with no signal observed at this size in CSK knockout cell line. To generate this image, wild-type and CSK knockout A549 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.

Intercept^® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBS was used as a blocking and diluting buffer.  

Lanes 1-4: Merged signal (red and green). Green - ab300132 observed at 50 kDa. Red - loading control ab8245 observed at 36 kDa. 

ab300132 Anti-CSK antibody [EPR24673-97] was shown to specifically react with CSK in wild-type HAP1 cells. Loss of signal was observed when knockout cell line was used. Wild-type and CSK knockout samples were subjected to SDS-PAGE. ab300132 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated at 4? overnight 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 10000 dilution for 1 hour at room temperature before imaging.

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

Exposure time: Lanes 1-3: 15 seconds; Lanes 4-7: 3 minutes; Lanes 8-9: 15 seconds. 

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

Immunohistochemical analysis of paraffin-embedded Human tonsil tissue labelling CSK with ab300132 at 1/5000 (0.111 µg/ml) followed by a ready to use LeicaDS9800 (BOND™ Polymer Refine Detection) was used. Positive staining on human tonsil (PMID: 9287362). The section was incubated with ab300132 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) was used.

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins

Immunohistochemical analysis of paraffin-embedded mouse spleen tissue labelling CSK with ab300132 at 1/5000 (0.111 µg/ml) followed by a ready to use LeicaDS9800 (BOND™ Polymer Refine Detection). Positive staining on mouse spleen. The section was incubated with ab300132 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: PBS was used instead of primary antibody (ab300132) followed by ready to use secondary antibody LeicaDS9800 (BOND™ Polymer Refine Detection) was used.

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins. 

Immunohistochemical analysis of paraffin-embedded rat spleen tissue labelling CSK with ab300132 at 1/5000 (0.111 µg/ml) followed by a ready to use LeicaDS9800 (BOND™ Polymer Refine Detection). Positive staining on rat spleen. The section was incubated with ab300132 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument Counterstained with Hematoxylin.

Secondary antibody only control: PBS was used instead of primary antibody (ab300132) followed by ready to use secondary antibody LeicaDS9800 (BOND™ Polymer Refine Detection).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins. 

Immunohistochemical analysis of paraffin-embedded human cardiac muscle tissue labelling CSK with ab300132 at 1/5000 (0.111 µg/ml) followed by a ready to use LeicaDS9800 (BOND™ Polymer Refine Detection) was used. Negative control: no staining on human cardiac muscle. The section was incubated with ab300132 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument.  Counterstained with Hematoxylin.

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins. 

Immunofluorescent analysis of 80% methanol-fixed, 0.1% TritonX-100 permeilized Jurkat (human T cell leukemia T lymphocyte) cells lebelling CSK with ab300132 at 1/50 (11.12 µg/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed antibody at 1/1000 dilution (Green). Confocal image showing cytoplasmic staining in Jurkat cell line. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 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 1/1000 dilution.

Intracellular flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized wild-type HAP1 (human chronic myelogenous leukemia near-haploid cell, Right) / CSK knockout HAP1(Left) cells labelling CSK with ab300132 at 1/500 dilution (0.1µg) (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 1/2000 dilution was used as the secondary antibody.

CSK was immunoprecipitated from 0.35 mg Ramos (human Burkitt's lymphoma b lymphocyte) whole cell lysate 10 µg with ab300132 at 1/30 dilution (2µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab300132 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 (Input): Ramos (human burkitt's lymphoma b lymphocyte) whole cell lysate 10 µg

Lane 2 (+): Ramos whole cell lysate

Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab300132 in Ramos whole cell lysate

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

Exposure time: 3 seconds

CSK was immunoprecipitated from 0.35 mg RAW 264.7 (mouse abelson murine leukemia virus-induced tumor macrophage) whole cell lysate 10 µg with ab300132 at 1/30 dilution (2µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab300132 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 (input): RAW 264.7 (mouse abelson murine leukemia virus-induced tumor macrophage) whole cell lysate 10 µg

Lane 2 (+): RAW 264.7 whole cell lysate

Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab300132 in RAW 264.7 whole cell lysate

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

Exposure time: 3 seconds

CSK was immunoprecipitated from 0.35 mg rat lymph node tissue lysate 10 µg with ab300132 at 1/30 dilution (2µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab300132 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 (input): Rat lymph node tissue lysate 10 µg

Lane 2 (+): Rat lymph node tissue lysate

Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab300132 in rat lymph node tissue lysate

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

Exposure time: 3 seconds. 

The identity of the lower MW band at approximately 37kDa is unknown.