Anti-Crk p38 antibody [22/Crk]

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-Crk p38 antibody [22/Crk] (AB300630)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized HeLa (human cervix adenocarcinoma epithelial cell) cells labelling Crk p38 with ab300630 at 1/50 (20ug/mL) dilution, followed by ab150117 Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 2 ug/mL dilution (Green). Confocal image showing cytoplasmic and nuclear staining in HeLa cell line is observed. ab179513 Anti-beta Tubulin rabbit monoclonal antibody was used to counterstain tubulin at 1/200 10 ug/mL dilution (Red). The Nuclear counterstain was DAPI (Blue). Secondary antibody only control : Secondary antibody is ab150117 Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 ug/mL dilution.

• IP

Supplier Data

Immunoprecipitation - Anti-Crk p38 antibody [22/Crk] (AB300630)

Crk p38 was immunoprecipitated from 0.35 mg HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate with ab300630 at 1/30 dilution (2 µg in 0.35 mg lysates). Western blot was performed on the immunoprecipitate using ab300630 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

This blot was developed using a high sensitivity ECL substrate. The high-sensitivity ECL substrate used allows for the detection of proteins in the mid-femtogram range.

ab300630 recognises a band at ~35 KDa (isoform Crk-II) and ~23KDa (isoform Crk-I).

All lanes:

Immunoprecipitation - Anti-Crk p38 antibody [22/Crk] (ab300630) at 1/1000 dilution

Lane 1:

HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate at 10 µg

Lane 2:

ab300630 at 1/30 IP in HeLa whole cell lysate at 10 µg

Lane 3:

Mouse IgG2a, kappa monoclonal isotype control (<a href='/en-us/products/primary-antibodies/mouse-igg2a-kappa-monoclonal-mopc-173-isotype-control-chip-grade-ab18413'>ab18413</a>) instead of ab300630 in HeLa whole cell lysate at 10 µg

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

Predicted band size: 33 kDa

Observed band size: 23 kDa,35 kDa

true

Exposure time: 3min

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-Crk p38 antibody [22/Crk] (AB300630)

Immunofluorescent analysis of 100% methanol-fixed, 0.1% TritonX-100 permeabilized C6 (rat glial tumor glial cell) cells labelling Crk p38 with ab300630 at 1/50 (20ug/mL) dilution, followed by ab150117 Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 2 ug/mL dilution (Green). Confocal image showing cytoplasmic and nuclear staining in C6 cell line is observed. ab179513 Anti-beta Tubulin rabbit monoclonal antibody was used to counterstain tubulin at 1/200 10 ug/mL dilution (Red). The Nuclear counterstain was DAPI (Blue). Secondary antibody only control : Secondary antibody is ab150117 Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 ug/mL dilution.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-Crk p38 antibody [22/Crk] (AB300630)

Immunofluorescent analysis of 100% methanol-fixed, 0.1% TritonX-100 permeabilized RAW 264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) cells labelling Crk p38 with ab300630 at 1/50 (20ug/mL) dilution, followed by ab150117 Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 2 ug/mL dilution (Green). Confocal image showing cytoplasmic and nuclear staining in RAW 264.7 cell line is observed. ab179513 Anti-beta Tubulin rabbit monoclonal antibody was used to counterstain tubulin at 1/200 10 ug/mL dilution (Red). The Nuclear counterstain was DAPI (Blue). Secondary antibody only control : Secondary antibody is ab150117 Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 ug/mL dilution.

• IP

Supplier Data

Immunoprecipitation - Anti-Crk p38 antibody [22/Crk] (AB300630)

Crk p38 was immunoprecipitated from 0.35 mg C6 (rat glial tumor glial cell) whole cell lysate with ab300630 at 1/30 dilution (2 µg in 0.35 mg lysates). Western blot was performed on the immunoprecipitate using ab300630 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

This blot was developed using a high sensitivity ECL substrate. The high-sensitivity ECL substrate used allows for the detection of proteins in the mid-femtogram range.

ab300630 recognises a band at ~35 KDa (isoform Crk-II) and ~23KDa (isoform Crk-I).

All lanes:

Immunoprecipitation - Anti-Crk p38 antibody [22/Crk] (ab300630) at 1/1000 dilution

Lane 1:

C6 (rat glial tumor glial cell) whole cell lysate at 10 µg

Lane 2:

ab300630 at 1/30 IP in C6 whole cell lysate at 10 µg

Lane 3:

Mouse IgG2a, kappa monoclonal isotype control (<a href='/en-us/products/primary-antibodies/mouse-igg2a-kappa-monoclonal-mopc-173-isotype-control-chip-grade-ab18413'>ab18413</a>) instead of ab300630 in C6 whole cell lysate at 10 µg

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

Predicted band size: 33 kDa

Observed band size: 23 kDa,35 kDa

true

Exposure time: 3min

• IP

Supplier Data

Immunoprecipitation - Anti-Crk p38 antibody [22/Crk] (AB300630)

Crk p38 was immunoprecipitated from 0.35 mg NIH/3T3 (mouse embryonic fibroblast) whole cell lysate with ab300630 at 1/30 dilution (2 µg in 0.35 mg lysates). Western blot was performed on the immunoprecipitate using ab300630 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

This blot was developed using a high sensitivity ECL substrate. The high-sensitivity ECL substrate used allows for the detection of proteins in the mid-femtogram range.

ab300630 recognises a band at ~35 KDa (isoform Crk-II) and ~23KDa (isoform Crk-I).

All lanes:

Immunoprecipitation - Anti-Crk p38 antibody [22/Crk] (ab300630) at 1/1000 dilution

Lane 1:

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

Lane 2:

ab300630 at 1/30 IP in NIH/3T3 whole cell lysate at 10 µg

Lane 3:

Mouse IgG2a, kappa monoclonal isotype control (<a href='/en-us/products/primary-antibodies/mouse-igg2a-kappa-monoclonal-mopc-173-isotype-control-chip-grade-ab18413'>ab18413</a>) instead of ab300630 in NIH/3T3 whole cell lysate at 10 µg

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

Predicted band size: 33 kDa

Observed band size: 23 kDa,35 kDa

true

Exposure time: 3min

• WB

Supplier Data

Western blot - Anti-Crk p38 antibody [22/Crk] (AB300630)

Blocking and diluting buffer and concentration : 5% NFDM/TBST The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID : 8943292). ab300630 recognises a band at ~35 KDa (isoform Crk-II) and ~23KDa (isoform Crk-I). Exposure time : Lanes 1-4 : 15 seconds Lanes 5-8 : 8 seconds

All lanes:

Western blot - Anti-Crk p38 antibody [22/Crk] (ab300630) at 1/1000 dilution

Lane 1:

HeLa (human cervix adenocarcinoma epithelial cell), whole cell lysate 10 µg

Lane 2:

K562 (human chronic myelogenous leukemia lymphoblast), whole cell lysate 10 µg

Lane 3:

HepG2 (human hepatocellar carcinoma epithelial cell), whole cell lysate 10 µg

Lane 4:

Ramos (human Burkitts lymphoma B lymphocyte), whole cell lysate 10 µg

Lane 5:

Jurkat (human T cell leukemia T lymphocyte), whole cell lysate 10 µg

Lane 6:

C6 (rat glial tumor glial cell), whole cell lysate 10 µg

Lane 7:

RAW264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage), whole cell lysate 10 µg

Lane 8:

PC-12 (rat adrenal gland pheochromocytoma), whole cell lysate 10 µg

Secondary

All lanes:

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

Observed band size: 35 kDa,23 kDa

false

• WB

Lab

Western blot - Anti-Crk p38 antibody [22/Crk] (AB300630)

Western blot : Anti-Crk p38 antibody [22/Crk] ab300630 staining at 1/1000 dilution, shown in green; Rabbit anti alpha Tubulin ab52866 loading control staining at 1/20000 dilution, shown in magenta. A band was observed at 35, 24 kDa in Wild-type HeLa cell lysates with no signal observed at this size in CRK knockout HeLa cell line. To generate this image, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 5pc 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 800CW and Goat anti-Rabbit 680RD at 1/20,000 dilution.

All lanes:

Western blot - Anti-Crk p38 antibody [22/Crk] (ab300630) at 1/1000 dilution

Lane 1:

Wild-type HeLa whole cell lysate at 20 µg

Lane 2:

Western blot - Human CRK (Crk p38) knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-crk-crk-p38-knockout-hela-cell-line-ab265097'>ab265097</a>) at 20 µg

Lane 3:

HepG2 whole cell lysate at 20 µg

Lane 4:

K562 whole cell lysate at 20 µg

Secondary

All lanes:

Goat anti-Mouse 800CW & Goat anti-Rabbit 680RD at 1/20000 dilution

Predicted band size: 34 kDa

Observed band size: 35 kDa,24 kDa

false