Anti-SHP2 抗体 [79/PTP1D/SHP2] (ab290646)

Blocking and diluting buffer and concentration: False colour image of Western blot: Anti-SHP2 antibody [79/PTP1D/SHP2] (ab290646 staining at 1/1000 dilution, shown in green; Rabbit anti-GAPDH antibody [16891] (ab181602) loading control staining at 1/20000 dilution, shown in red.

In Western blot, ab290646 was shown to bind specifically to SHP2(PTPN11). A band was observed at 68 kDa in wild-type HEK-293 cell lysates with no signal observed at this size in the PTPN11 knockout cell line. To generate this image, wild-type and PTPN11 knockout HEK-293 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a PVDF-FL membrane. Membranes were blocked in Odyssey diluted in equal volume of 0.1 % TBS 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 (IRDye^® 800CW) (ab216772) and Goat Anti-Rabbit IgG H&L (IRDye^® 680RD) (ab216777) at 1/10000 dilution.

Performed under reducing conditions.

Blocking and diluting buffer and concentration: 5% NFDM/TBSTLysates should be made freshly and used in WB immediately to minimize protein degradation.

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

Exposure time: Lanes 1/2: 37 seconds Lanes 3/4: 8 seconds

Immunohistochemical analysis of paraffin-embedded Human ovarian cancer tissue labelling SHP2 with AB290646 at 1/500 (2.018 ug/ml) followed by a ready to use LeicaDS9800 (Bond Polymer Refine Detection) was used. Positive staining in human ovarian cancer.The section was incubated with ab290646 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.

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 solution2) for 20 mins

Immunohistochemical analysis of paraffin-embedded Mouse kidney tissue labelling SHP2 with AB290646 at 1/2000 (0.505 ug/ml) followed by a ready to use LeicaDS9800 (Bond Polymer Refine Detection) was used. Positive staining in mouse kidney.The section was incubated with ab290646 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.

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 solution2) for 20 mins

Immunohistochemical analysis of paraffin-embedded Rat kidney tissue labelling SHP2 with AB290646 at 1/2000 (0.505 ug/ml) followed by a ready to use LeicaDS9800 (Bond Polymer Refine Detection) was used. Positive staining in rat kidney.The section was incubated with ab290646 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.

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 solution2) for 20 mins

Immunohistochemical analysis of paraffin-embedded Human liver tissue labelling SHP2 with AB290646 at 1/500 (2.018 µg/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used. Negative control: no staining in human liver (PMID: 8216283).The section was incubated with ab290646 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.

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 solution2) for 20 mins

Immunohistochemical analysis of paraffin-embedded Mouse cerebrum tissue labelling SHP2 with AB290646 at 1/2000 (0.505 ug/ml) followed by a ready to use LeicaDS9800 (Bond Polymer Refine Detection) was used. Positive staining in mouse cerebrum.The section was incubated with ab290646 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.

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 solution2) for 20 mins

Immunohistochemical analysis of paraffin-embedded Rat cerebrum tissue labelling SHP2 with AB290646 at 1/2000 (0.505 ug/ml) followed by a ready to use LeicaDS9800 (Bond Polymer Refine Detection) was used. Positive staining in rat cerebrum.The section was incubated with ab290646 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.

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 solution2) for 20 mins

Immunofluorescent analysis of 80% Methanol-fixed, 0.1% TritonX-100 permeabilized Jurkat (human T cell leukemia T lymphocyte) cells labeling SHP2 with AB290646 at 1/50 (20.18 µg/ml) dilution, followed by ab150117 Goat Anti-Mouse IgG H&L (Alexa Fluor^® 488) preadsorbed antibody at 1/1000 (2 µg/ml) dilution (Green). Confocal image showing cytoplasmic staining in Jurkat cell line is observed. ab179513 Anti-beta Tubulin rabbit monoclonal antibody was used to counterstain tubulin at 1/200 10 µg/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 µg/ml dilution.

SHP2 was immunoprecipitated from 0.35 mg HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate 10ug with ab290646 at 1/30 dilution (2µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab290646 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

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

Lane 2: ab290646 IP in HeLa whole cell lysate

Lane 3: Mouse IgG1 monoclonal (ab18443) instead of ab290646 in HeLa whole cell lysate

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

Exposure time: 3 seconds

SHP2 was immunoprecipitated from 0.35 mg NIH/3T3 (mouse embryonic fibroblast) whole cell lysate 10 µg with ab290646 at 1/30 dilution (2µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab290646 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 10 µg

Lane 2: ab290646 IP in NIH/3T3 whole cell lysate

Lane 3: Mouse IgG1 monoclonal (ab18443) instead of ab290646 in NIH/3T3 whole cell lysate

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

Exposure time: 3 seconds