Anti-CD3 zeta (phospho Y142) antibody [EP265(2)Y]

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-CD3 zeta (phospho Y142) antibody [EP265(2)Y] (AB68235)

Immunocytochemistry/ Immunofluorescence analysis of Jurkat (Human T cell leukemia T lymphocyte) treated with 1mM pervanadate for 30 minutes cells labeling CD3 zeta with purified ab68235 at 1/500 dilution (0.2 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) at 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 (blue) was used as nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-CD3 zeta (phospho Y142) antibody [EP265(2)Y] (AB68235)

Intracellular Flow Cytometry analysis of Jurkat (Human T cell leukemia T lymphocyte) treated with 50nM pervanadate for 5 minutes cells labeling CD3 zeta with purified ab68235 at 1/120 dilution (1μg/ml) (red). Cells were fixed with 4% Paraformaldehyde and permeabilised with 90% Methanol. A Goat anti rabbit IgG (Alexa Fluorr^® 488, ab150077) secondary antibody was used at 1/2000. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue). Untreated Jurkat cells (Green).

• IP

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Immunoprecipitation - Anti-CD3 zeta (phospho Y142) antibody [EP265(2)Y] (AB68235)

ab68235 (purified) at 1/20 dilution (0.5ug) immunoprecipitating CD3 zeta in Jurkat treated with 50nM pervanadate for 5 minutes whole cell lysate whole cell lysate. Jurkat (Human T cell leukemia T lymphocyte) treated with 50nM pervanadate for 5 minutes whole cell lysate 10ug
Lane 2 (+) : ab68235 & Jurkat treated with 50nM pervanadate for 5 minutes whole cell lysate whole cell lysate
Lane 3 (-) : Rabbit monoclonal IgG (ab172730) instead of ab68235 in Jurkat treated with 50nM pervanadate for 5 minutes whole cell lysate
For western blotting, VeriBlot for IP secondary antibody (HRP) (ab131366) was used at 1/2000 dilution.
Blocking and diluting buffer : 5% NFDM/TBST.

All lanes:

Immunoprecipitation - Anti-CD3 zeta (phospho Y142) antibody [EP265(2)Y] (ab68235)

Predicted band size: 19 kDa

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• WB

Lab

Western blot - Anti-CD3 zeta (phospho Y142) antibody [EP265(2)Y] (AB68235)

All lanes:

Western blot - Anti-CD3 zeta (phospho Y142) antibody [EP265(2)Y] (ab68235) at 1/1000 dilution

Lane 1:

Jurkat (Human T cell leukemia T lymphocyte) whole cell lysates at 15 µg

Lane 2:

Jurkat (Human T cell leukemia T lymphocyte) treated with 50mM pervanadate for 5 minutes whole cell lysates 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: 19 kDa

Observed band size: 19 kDa

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