44-554G

antibody from Invitrogen Antibodies

Targeting: PTPN11 BPTP3, NS1, PTP2C, SH-PTP2, SHP-2, SHP2

Western blot (Supportive data in Antibodypedia)

Immunocytochemistry (Supportive data in Antibodypedia)

Immunohistochemistry (Supportive data in Antibodypedia)

Antibody Data

Product number

44-554G

Provider

Invitrogen Antibodies

Product name

Phospho-SHP2 (Tyr542) Polyclonal Antibody

Antibody type

Polyclonal

Antigen

Synthetic peptide

Reactivity

Human, Mouse

Host

Rabbit

Isotype

IgG

Vial size

100 µL

Storage

-20°C

Western blot

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Peptide Competition. Lysates prepared from NIH3T3 cells left untreated (1) or treated with PDGF (2-5). were resolved by SDS-PAGE on a 10% polyacrylamide gel and transferred to PVDF. Membranes were blocked with a 3% BSA-TBST buffer for one hour at room temperature, and incubated with SHP2 (pY542) antibody for two hours at room temperature in a 3% BSA-TBST buffer, following prior incubation with: no peptide (1, 2), the non-phosphopeptide corresponding to the immunogen (3), a generic phosphotyrosine-containing peptide (4), or, the phosphopeptide immunogen (5). After washing, membranes were incubated with goat F (ab’)2 anti-rabbit IgG HRP conjugate (Product # ALI4404) and bands were detected using the Pierce SuperSignal™ method. The data show that only the peptide corresponding to SHP2 (pY542) blocks the signal, verifying the specificity of the antibody.

Immunocytochemistry

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunofluorescence analysis of Phospho-SHP-2 (Tyr542) Polyclonal Antibody was performed using 90% confluent log phase A-431 cells treated with 200 ng/mL of EGF for 10 minutes. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled with Phospho-SHP-2 (Tyr542) Polyclonal Antibody (Product # 44-554G) at 1:250 dilution in 0.1% BSA and incubated overnight at 4 degree and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034) at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). F-actin (Panel c: red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing membrane localization. Panel e represents cells treated with antagonist, Afatinib (0.5 uM for 6hrs) followed by EGF (200 ng/mL for 10 minutes), showing reduced expression of phospho-SHP2. Panel f shows untreated cells with no signal. Panel g represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

Immunohistochemistry

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunohistochemistry analysis of Phospho-SHP-2/PTPN11 showing staining in the cytoplasm of paraffin-embedded human brain tissue (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with a Anti- Phospho-SHP-2/PTPN11 pSer542 Polyclonal Antibody (Product # 44-554G) diluted in 3% BSA-PBS at a dilution of1:20 overnight at 4°C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.