Anti-CSK antibody [EPR24673-97]

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CSK antibody [EPR24673-97] (AB300132)

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

• ICC

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Immunocytochemistry - Anti-CSK antibody [EPR24673-97] (AB300132)

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.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-CSK antibody [EPR24673-97] (AB300132)

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.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CSK antibody [EPR24673-97] (AB300132)

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.

• IP

Supplier Data

Immunoprecipitation - Anti-CSK antibody [EPR24673-97] (AB300132)

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.

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

All lanes:

Immunoprecipitation - Anti-CSK antibody [EPR24673-97] (ab300132) at 1/1000 dilution

Lane 1:

Ramos (human burkitt's lymphoma b lymphocyte) whole cell lysate at 10 µg

Lane 2:

ab300132 at 1/30 IP in Ramos whole cell lysate

Lane 3:

Rabbit monoclonal IgG (<a href='/en-us/products/primary-antibodies/rabbit-igg-monoclonal-epr25a-isotype-control-ab172730'>ab172730</a>) instead of ab300132 in Ramos whole cell lysate

Observed band size: 50 kDa

false

Exposure time: 3s

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CSK antibody [EPR24673-97] (AB300132)

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.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CSK antibody [EPR24673-97] (AB300132)

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.

• IP

Supplier Data

Immunoprecipitation - Anti-CSK antibody [EPR24673-97] (AB300132)

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.

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

All lanes:

Immunoprecipitation - Anti-CSK antibody [EPR24673-97] (ab300132) at 1/1000 dilution

Lane 1:

RAW 264.7 (mouse abelson murine leukemia virus-induced tumor macrophage) whole cell lysate at 10 µg

Lane 2:

ab300132 at 1/30 IP in RAW 264.7 whole cell lysate at 10 µg

Lane 3:

Rabbit monoclonal IgG (<a href='/en-us/products/primary-antibodies/rabbit-igg-monoclonal-epr25a-isotype-control-ab172730'>ab172730</a>) instead of ab300132 in RAW 264.7 whole cell lysate at 10 µg

Observed band size: 50 kDa

false

Exposure time: 3s

• IP

Supplier Data

Immunoprecipitation - Anti-CSK antibody [EPR24673-97] (AB300132)

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.

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

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

All lanes:

Immunoprecipitation - Anti-CSK antibody [EPR24673-97] (ab300132) at 1/1000 dilution

Lane 1:

Rat lymph node tissue lysate at 10 µg

Lane 2:

ab300132 in Rat lymph node tissue lysate at 10 µg

Lane 3:

Rabbit monoclonal IgG (<a href='/en-us/products/primary-antibodies/rabbit-igg-monoclonal-epr25a-isotype-control-ab172730'>ab172730</a>) instead of ab300132 in rat lymph node tissue lysate at 10 µg

Observed band size: 50 kDa

false

Exposure time: 3s

• WB

Lab

Western blot - Anti-CSK antibody [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.

All lanes:

Western blot - Anti-CSK antibody [EPR24673-97] (ab300132) at 1/1000 dilution

Lane 1:

Wild-type A549 cell lysate at 20 µg

Lane 2:

CSK knockout A549 cell lysate at 20 µg

Lane 2:

Western blot - Human CSK knockout A549 cell line (<a href='/en-us/products/cell-lines/human-csk-knockout-a549-cell-line-ab300902'>ab300902</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: 50 kDa

false

• WB

Supplier Data

Western blot - Anti-CSK antibody [EPR24673-97] (AB300132)

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

All lanes:

Western blot - Anti-CSK antibody [EPR24673-97] (ab300132) at 1/1000 dilution

Lane 1:

Human tonsil tissue lysate at 20 µg

Lane 2:

Mouse spleen 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: 50 kDa

false

Exposure time: 3s

• WB

Supplier Data

Western blot - Anti-CSK antibody [EPR24673-97] (AB300132)

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°C 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.

All lanes:

Western blot - Anti-CSK antibody [EPR24673-97] (ab300132) at 1/1000 dilution

Lane 1:

Wild-type HAP1 whole cell lysate at 20 µg

Lane 2:

CSK knockout HAP1 whole cell lysate at 20 µg

Lane 3:

Jurkat (Human T cell leukemia cell line from peripheral blood) whole cell lysate at 20 µg

Lane 4:

C6 (Rat glial tumor glial cell) whole cell lysate at 20 µg

Secondary

All lanes:

Intercept® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBS at 1/10000 dilution

Observed band size: 50 kDa

false

• WB

Supplier Data

Western blot - Anti-CSK antibody [EPR24673-97] (AB300132)

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.

All lanes:

Western blot - Anti-CSK antibody [EPR24673-97] (ab300132) at 1/1000 dilution

Lane 1:

Ramos (Human burkitt's lymphoma b lymphocyte) whole cell lysate at 1/20 dilution

Lane 2:

Jurkat (Human T cell leukemia cell line from peripheral blood) whole cell lysate at 1/20 dilution

Lane 3:

RAW 264.7 (Mouse abelson murine leukemia virus-induced tumor macrophage) whole cell lysate at 1/20 dilution

Lane 4:

NIH/3T3 (Mouse embryonic fibroblast) whole cell lysate at 1/20 dilution

Lane 5:

C6 (Rat glial tumor glial cell) whole cell lysate at 1/20 dilution

Lanes 6 - 7:

PC-12 (Rat adrenal gland pheochromocytoma) whole cell lysate at 1/20 dilution

Lane 8:

Mouse lymph node tissue lysate at 1/20 dilution

Lane 9:

Rat lymph node tissue lysate at 1/20 dilution

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 1/50000 dilution

Observed band size: 50 kDa

false