Anti-SHP2 (phospho Y542) antibody

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SHP2 (phospho Y542) antibody (AB17939)

Immunohistochemical analysis of paraffin-embedded human brain labeling SHP2 (phospho Y542) with ab17939 at 1/20 dilution (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 ab17939 diluted in 3% BSA-PBS at a dilution of 1/20 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.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-SHP2 (phospho Y542) antibody (AB17939)

Immunofluorescence analysis of 90% confluent log phase A-431 (Human epidermoid carcinoma cell line) cells treated with 0.2 ug/mL of EGF for 10 minutes labeling SHP2 (phospho Y542) with ab17939 at 1/250 dilution.

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 ab17939 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 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. F-actin (Panel c : red) was stained with Rhodamine Phalloidin at 1/300 dilution. 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 (0.2 ug/mL for 10 minutes), showing reduced expression of SHP2 (phospho Y542). 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.

• WB

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Western blot - Anti-SHP2 (phospho Y542) antibody (AB17939)

Western blot using ab17939 on 10-30μg NIH3T3 cell lysate. Lane 1 : untreated cells. Lane 2 : cells treated with PDGF. Lane 3 : cells treated with PDGF. Antibody blocked with non-phosphorylated immunopeptide. Lane 4 : cells treated with PDGF. Antibody blocked with a generic tyrosine-phosphorylated peptide. Lane 5 : cells treated with PDGF. Antibody blocked with phosphorylated immunopeptide.

Western blot using ab17939 on 10-30µg NIH3T3 cell lysate. Lane 1 : untreated cells.
Lane 2 : cells treated with PDGF.
Lane 3 : cells treated with PDGF. Antibody blocked with non-phosphorylated immunopeptide.
Lane 4 : cells treated with PDGF. Antibody blocked with a generic tyrosine-phosphorylated peptide.
Lane 5 : cells treated with PDGF. Antibody blocked with phosphorylated immunopeptide.

All lanes:

Western blot - Anti-SHP2 (phospho Y542) antibody (ab17939)

Predicted band size: 68 kDa

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