Invitrogen

Phospho-SHP2 (Tyr542) Polyclonal Antibody

Advanced Verification

This Antibody was verified by Cell treatment to ensure that the antibody binds to the antigen stated.

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Datasheet

Protocols

Questions & Answers

Tech Support

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Phospho-SHP2 (Tyr542) Antibody in Immunocytochemistry (ICC/IF) — 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.

Phospho-SHP2 (Tyr542) Antibody in Immunohistochemistry (Paraffin) (IHC (P)) — 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.

Phospho-SHP2 (Tyr542) Antibody in Western Blot (WB) — 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.

Phospho-SHP2 (Tyr542) Antibody — Altered expression of target protein upon cell treatment demonstrates antibody specificity. Immunofluorescence analysis of Phospho-SHP-2 (Tyr542) using Phospho-SHP-2 (Tyr542) Polyclonal Antibody (Product # 44-554G) shows induction of SHP-2 phosphorylation at Tyr542 residue in A-431 cell line upon EGF treatment. Pre-treatment with EGFR-antagonist Afatinib resulted in inhibition of phosphorylation following EGF treatment.

Product Details

44-554G

Applications

Tested Dilution

Publications

Western Blot (WB)

1:1,000

Immunohistochemistry (Paraffin) (IHC (P))

1:20

Immunocytochemistry (ICC/IF)

5 µg/mL

Product Specifications

Species Reactivity

Human, Mouse

Host/Isotype

Rabbit / IgG

Class

Polyclonal

Type

Antibody

Immunogen

The antiserum was produced against a chemically synthesized phosphopeptide derived from the region of human SHP2 that contains tyrosine 542. The sequence is conserved in mouse, rat and chicken.

View immunogen

Conjugate

Unconjugated

Form

Liquid

Purification

Antigen affinity chromatography

Storage buffer

Dulbecco's PBS, pH 7.3, with 1mg/mL BSA

Contains

0.05% sodium azide

Storage conditions

-20°C

RRID

AB_2533679

Target Information

The protein encoded by this gene is a member of the protein tyrosine phosphatase (PTP) family. PTPs are known to be signaling molecules that regulate a variety of cellular processes including cell growth, differentiation, mitotic cycle, and oncogenic transformation. This PTP contains two tandem Src homology-2 domains, which function as phospho-tyrosine binding domains and mediate the interaction of this PTP with its substrates. This PTP is widely expressed in most tissues and plays a regulatory role in various cell signaling events that are important for a diversity of cell functions, such as mitogenic activation, metabolic control, transcription regulation, and cell migration. Mutations in this gene are a cause of Noonan syndrome as well as acute myeloid leukemia.

For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.

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Bioinformatics

Protein Aliases: HCP; HGNC:9644; MGC14433; Noonan syndrome 1; OTTHUMP00000166108; Protein-tyrosine phosphatase 1D; Protein-tyrosine phosphatase 2C; Protein-tyrosine phosphatase SYP; PTP-1D; PTP-2C; PTP1C; SH-PTP2; SH-PTP3; SH2 domain-containing protein tyrosine phosphatase-2; SHP-2; Tyrosine-protein phosphatase non-receptor type 11

Gene Aliases: 2700084A17Rik; AW536184; BPTP3; CFC; JMML; METCDS; NS1; PTP-1D; PTP1D; PTP2C; PTPN11; SAP-2; SH-PTP2; SH-PTP3; SHP-2; SHP2; SHPTP2; Syp

UniProt ID: (Human) Q06124, (Mouse) P35235

Entrez Gene ID: (Human) 5781, (Mouse) 19247

Molecular Function: protein phosphatase