All lanes : Anti-SHP2 antibody [Y478] (ab32083) at 1/1000 dilution

Lane 1 : Wild-type HEK-293T cell lysate
Lane 2 : PTPN11 knockout HEK-293T cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 68 kDa
Observed band size: 68 kDa

Lanes 1- 2: Merged signal (red and green). Green - ab32083 observed at 68 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

 ab32083 was shown to react with SHP2 in wild-type HEK-293T cells in western blot. Loss of signal was observed when knockout cell line ab266450 (knockout cell lysate ab257618) was used. Wild-type HEK-293T and PTPN11 knockout HEK-293T cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab32083 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 1000 dilution and a 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 20000 dilution for 1 hour at room temperature before imaging.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human endometrium cancer tissue sections labeling SHP2 with Purified ab32083 at 1:100 dilution (5.51 µg/ml). Heat mediated antigen retrieval was performed using Perform heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0)ImmunoHistoProbe one step HRP Polymer (ready to use)was used as the secondary antibody.Negative control:PBS instead of the primary antibody.Hematoxylinwas used as a counterstain

Immunocytochemistry/ Immunofluorescence analysis of A431 (Human epidermoid carcinoma epithelial cell) cells labeling SHP2 with Purified ab32083 at 1:50 dilution (11 µg/ml). Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. Cells were counterstained with Ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor®594) 1:200 (2.5 µg/ml). Goat anti rabbit IgG (Alexa Fluor®488, ab150077) was used as the secondary antibody at 1:1000 (2 µg/ml) dilution. DAPI nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

Flow Cytometry analysis of Jurkat (Human T cell leukemia T lymphocyte) cells labeling SHP2 with purified ab32083 at 1:50 dilution (10 µg/ml) (red). Cells were fixed with 4% Paraformaldehyde. A Goat anti rabbit IgG (Alexa Fluor® 488) secondary antibody was used at 1:2000 dilution. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).

Lane 1: Wild-type HAP1 cell lysate (20 µg)
Lane 2: SHP2 knockout HAP1 cell lysate (20 µg)
Lane 3: A431 cell lysate (20 µg)
Lane 4: Jurkat cell lysate (20 µg)
Lanes 1 to 4: Merged signal (red and green). Green - ab32083 observed at 68 kDa. Red - loading control, ab8245, observed at 37 kDa.
unpurified ab32083 was shown to specifically react with SHP2 when SHP2 knockout samples were used. Wild-type and SHP2 knockout samples were subjected to SDS-PAGE. ab32083 and ab8245 (loading control to GAPDH) were both diluted 1/1000 and 1/10 000 respectively and incubated overnight at 4°C. 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 1/10 000 dilution for 1 h at room temperature before imaging.

Anti-SHP2 antibody [Y478] (ab32083) at 0.3 µg/ml (purified) + THP-1 (Human monocytic leukemia monocyte) whole cell lysates at 15 µg

Secondary
Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 68 kDa

Blocking and diluting buffer: 5% NFDM/TBST. 

ab32083 (purified) at 1:40 dilution (2µg) immunoprecipitating SHP2 in THP-1 whole cell lysate.
Lane 1 (input): THP-1 (Human monocytic leukemia monocyte) whole cell lysate 10µg
Lane 2 (+): ab32083 & THP-1 whole cell lysate
Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab32083 in THP-1 whole cell lysate
For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366) was used for detection at 1:1000 dilution.
Blocking and diluting buffer: 5% NFDM/TBST.

Overlay histogram showing HAP1 wildtype (green line) and HAP1-PTPN11 knockout cells (red line) stained with ab32083. The cells were fixed with 4% formaldehyde (10 min) and then permeabilized with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS / 10% normal goat serum to block non-specific protein-protein interactions followed by the antibody (unpurified ab32083, 0.1µg/ml) for 30 min at 22°C. The secondary antibody used was Alexa Fluor® 488 goat anti-rabbit IgG (H&L) presorbed (ab150081) at 1/2000 dilution for 30 min at 22°C.

A rabbit IgG isotype control antibody  (ab172730) was used at the same concentration and conditions as the primary antibody (HAP1 wildtype - black line, HAP1-PTPN11  knockout - grey line). Unlabelled sample was also used as a control (this line is not shown for the purpose of simplicity).

Acquisition of >5,000 events were collected using a 50 mW Blue laser (488nm) and 530/30 bandpass filter.

This antibody can also be used in HAP1 cells fixed with 80% methanol (5 min) permeabilized with 0.1% PBS-Triton X-100 for 15 min under the same conditions.

Flow Cytometry analysis of Jurkat (human acute T cell leukemia) cells labeling SHP2 with unpurified ab32083 at 1:500 dilution(1ug/ml) (red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. A Goat anti rabbit IgG (Alexa Fluor® 488)(ab150077)(1:2000 dilution) was used as the secondary antibody. Rabbit monoclonal IgG (Black)(ab172730) was used as the isotype control, Cell without incubation with primary antibody and secondary antibody (Blue) were used as the unlabeled control.

Immunohistochemical analysis of SHP2 expression in paraffin embedded human breast carcinoma, using 1/50 unpurified ab32083.

unpurified ab32083 stained Hek293 cells. The cells were 100% methanol fixed for 5 minutes at -20°C and then incubated in 1%BSA / 10% normal goat  serum / 0.3M glycine in 0.1% PBS-Tween for 1hour at room temperature to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab32083 at 1/100 dilution) overnight at +4°C. The secondary antibody (pseudo-colored green) was Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed (ab150081) used at a 1/1000 dilution for 1hour at room temperature. Alexa Fluor® 594 WGA was used to label plasma membranes (pseudo-colored red) at a 1/200 dilution for 1hour at room temperature. DAPI was used to stain the cell nuclei (pseudo-colored blue) at a concentration of 1.43µM for 1hour at room temperature.

Anti-SHP2 antibody [Y478] (ab32083) at 1/5000 dilution (unpurified) + Jurkat cell lysate

Predicted band size: 68 kDa
Observed band size: 70 kDa why is the actual band size different from the predicted?