Anti-SHP2 (phospho Y542) antibody [EP508(2)Y]

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-SHP2 (phospho Y542) antibody [EP508(2)Y] (AB62322)

ab62322 staining SHP2 in THP-1 cells ( Human monocytic leukaemia cell line) by ICC/IF (immunocytochemistry/immunofluorescence). Cells were fixed with 4% Paraformaldehyde and permeabilized with 0.1% TritonX-100. Samples were incubated with purified ab62322 at 8.8 μg/ml. Secondary antibody used was AlexaFluor^®488 Goat anti-Rabbit (ab150077) at 2 μg/ml. Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594)( ab195889) used as counterstain at 2.5 μg/ml . DAPI was used as nuclear counterstain. Confocal image showing the expression was increased after treatment with TPA 200nM for 24h and PDGF 50ng/ml for 30min, the signal decreased after treatment with Lambda Protein Phosphatase 31℃ for 2h.

This image was generated using the unpurified version of the product.

• IP

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Immunoprecipitation - Anti-SHP2 (phospho Y542) antibody [EP508(2)Y] (AB62322)

ab62322 (purified) at 1 : 30 dilution (2μg) immunoprecipitating SHP2 in NIH/3T3 treated with 50ng/ml PDGF for 40min whole cell lysate.
Lane 1 (input) : NIH/3T3 (Mouse embryonic fibroblast) treated with 50ng/ml PDGF for 40min whole cell lysate 10μg
Lane 2 (+) : ab62322 & NIH/3T3 treated with 50ng/ml PDGF for 40min whole cell lysate
Lane 3 (-) : Rabbit monoclonal IgG (ab172730) instead of ab62322 in NIH/3T3 treated with 50ng/ml PDGF for 40min 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.

All lanes:

Immunoprecipitation - Anti-SHP2 (phospho Y542) antibody [EP508(2)Y] (ab62322)

Predicted band size: 68 kDa

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• WB

Lab

Western blot - Anti-SHP2 (phospho Y542) antibody [EP508(2)Y] (AB62322)

Blocking and diluting buffer used was 5% NFDM/TBST.

Purified batch of ab62322 was used.

This image was generated using the unpurified version of the product.

All lanes:

Western blot - Anti-SHP2 (phospho Y542) antibody [EP508(2)Y] (ab62322) at 1/200 dilution

Lane 1:

THP-1 (Human monocytic leukemia monocyte) whole cell lysates at 15 µg

Lane 2:

THP-1 (Human monocytic leukemia monocyte) treated with Phorbol-12-myristate-13-acetate and platelet-derived growth factor at 15 µg

Lane 3:

THP-1 (Human monocytic leukemia monocyte) treated with Phorbol-12-myristate-13-acetate and platelet-derived growth factor, then the membrane was incubated with phosphatase at 15 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 68 kDa

Observed band size: 68 kDa

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• WB

Unknown

Western blot - Anti-SHP2 (phospho Y542) antibody [EP508(2)Y] (AB62322)

Beta Tubulin has been included as a loading control.

This image was generated using the unpurified version of the product.

All lanes:

Western blot - Anti-SHP2 (phospho Y542) antibody [EP508(2)Y] (ab62322) at 1/50000 dilution

Lane 1:

NIH/3T3 cell lysates; untreated at 10 µg

Lane 2:

NIH/3T3 cell lysates; treated with PDGF at 10 µg

Secondary

All lanes:

goat anti-rabbit HRP conjugated, at 1/2000 dilution

Predicted band size: 68 kDa

Observed band size: 68 kDa

false

• WB

Lab

Western blot - Anti-SHP2 (phospho Y542) antibody [EP508(2)Y] (AB62322)

All lanes:

Western blot - Anti-SHP2 (phospho Y542) antibody [EP508(2)Y] (ab62322) at 1/1000 dilution

Lane 1:

NIH/3T3 (Mouse embryonic fibroblast) whole cell lysates at 15 µg

Lane 2:

NIH/3T3 (Mouse embryonic fibroblast) treated with 40ng/ml PDGF for 40 minutes whole cell lysates at 15 µg

Lane 3:

NIH/3T3 (Mouse embryonic fibroblast) treated with 40ng/ml PDGF for 40 minutes then incubated with phosphatase at 15 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 68 kDa

Observed band size: 68 kDa

false