Anti-SHP2 抗体 [EPR26539-45] (ab300579)

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

Lysates at 20 µg per lane.

In Western blot, ab300579 was shown to bind specifically to PTPN11 (SHP2). A band was observed at 72 kDa in wild-type HEK293T cell lysates with no signal observed at this size in PTPN11 (SHP2) knockout cell line ab266450 (knockout cell lysate ab257618). 

Exposure time: 180 seconds

Blocking and diluting 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.

The bands beneath the target band (72 kDa) are likely to be degraded target fragments.

Exposure time: 26 seconds

Blocking and diluting 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.

The bands beneath the target band (72 kDa) are likely to be degraded target fragments.

Exposure time: 81 seconds

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

Lysate was freshly made and used for Western blotting immediately to minimize protein degradation.

Exposure time: 48 seconds

Immunohistochemical analysis of paraffin-embedded human tonsil tissue labeling SHP2 with ab300579 at 1/50 (9.76 µg/mL) followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP polymer). Positive staining in human tonsil (PMID:18728972). The section was incubated with ab300579 at 4°C overnight. Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP polymer).

Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0)

Immunohistochemical analysis of paraffin-embedded mouse testis tissue labeling SHP2 with ab300579 at 1/50 (9.76 µg/mL) followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP polymer). Positive staining in mouse testis (PMID:24123360). The section was incubated with ab300579 at 4°C overnight. Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP polymer).

Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0)

Immunohistochemical analysis of paraffin-embedded rat testis tissue labeling SHP2 with ab300579 at 1/50 (9.76 µg/mL) followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP polymer). Positive staining in rat testis (PMID:24123360). The section was incubated with ab300579 at 4°C overnight. Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP polymer).

Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0)

Immunohistochemical analysis of paraffin-embedded (A) Wild-type HEK293 tissue labeling SHP2 with ab300579 at 1/200 (1.22 µg/mL) followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP polymer). Positive staining in wild-type HEK293T cells and no staining in PTPN11 (SHP2) knockout HEK293T cells. The section was incubated with ab300579 at 4°C overnight. Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP polymer).

Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0)

SHP2 was immunoprecipitated from 0.35 mg NIH/3T3 (mouse embryonic fibroblast), whole cell lysate 10µg with ab300579 at 1/30 dilution (2µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab300579 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: NIH/3T3 (mouse embryonic fibroblast), whole cell lysate 10µg

Lane 2: ab300579 IP in NIH/3T3 whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab300579 in NIH/3T3 whole cell lysate

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

Exposure time: 180 seconds

SHP2 was immunoprecipitated from 0.35 mg HeLa (human cervical adenocarcinoma epithelial cell), whole cell lysate 10 µg with ab300579 at 1/30 dilution (2µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab300579 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: HeLa (human cervical adenocarcinoma epithelial cell), whole cell lysate 10 µg

Lane 2: ab300579 IP in Hela whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab300579 in Hela whole cell lysate

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

Exposure time: 180 seconds

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized PTPN11 KO HEK293T (PTPN11 KO knock out human embryonic kidney epithelial cell) (ab266450) cells labeling SHP2 with ab300579 at 1/50 (9.76 µg/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed antibody at 1/1000 (2 µg/ml) dilution (Green). Confocal image showing cytoplasmic and weak nuclear staining in wildtype HEK293T cell line, while showing no staining in PTPN11 knockout HEK293T cell line is observed. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 (2.5 µg/ml) 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 (2 µg/ml) dilution.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized NIH/3T3 (mouse embryonic fibroblast) cells labeling SHP2 with ab300579 at 1/50 (9.76 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed antibody at 1/1000 (2 µg/ml) dilution (Green). Confocal image showing cytoplasmic and weak nuclear staining in NIH/3T3 cell line is observed. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 (2.5 µg/ml) 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 (2 µg/ml) dilution.