Anti-CD3 zeta (phospho Y142) antibody [EP265(2)Y] - BSA and Azide free

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-CD3 zeta (phospho Y142) antibody [EP265(2)Y] - BSA and Azide free (AB245765)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab68235).

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-CD3 zeta (phospho Y142) antibody [EP265(2)Y] - BSA and Azide free (AB245765)

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 Fluor^® 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).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab68235).

• IP

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Immunoprecipitation - Anti-CD3 zeta (phospho Y142) antibody [EP265(2)Y] - BSA and Azide free (AB245765)

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.
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab68235).

All lanes:

Immunoprecipitation - Anti-CD3 zeta (phospho Y142) antibody [EP265(2)Y] (<a href='/en-us/products/primary-antibodies/cd3-zeta-phospho-y142-antibody-ep2652y-ab68235'>ab68235</a>)

Predicted band size: 19 kDa

false

• WB

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Western blot - Anti-CD3 zeta (phospho Y142) antibody [EP265(2)Y] - BSA and Azide free (AB245765)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab68235).

All lanes:

Western blot - Anti-CD3 zeta (phospho Y142) antibody [EP265(2)Y] - BSA and Azide free (ab245765) 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

false