Anti-CD3 zeta (phospho Y83) antibody [EP776(2)Y]

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-CD3 zeta (phospho Y83) antibody [EP776(2)Y] (AB68236)

Flow Cytometry analysis of Jurkat (human acute T cell leukemia) treated (Red)/untreated (Green) with 1mM pervanadate for 4 hours with purified ab68236 at 1/250 dilution. The secondary antibody was Goat anti rabbit IgG (Alexa Fluor^® 488) at 1/2000 dilution. A Rabbit monoclonal IgG (Black) was used as the isotype control and cells without incubation with primary antibody and secondary antibody (Blue) were used as unlabeled control.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-CD3 zeta (phospho Y83) antibody [EP776(2)Y] (AB68236)

Immunocytochemistry/Immunofluorescence analysis of Jurkat cells (untreated, Per treated and Per+LP treated) labelling CD3 zeta (phospho Y83) with ab68236 (left) and CD3 zeta with ab40804 (right) both at a dilution of 1/200. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody. DAPI (blue) was used as the nuclear counterstain. ab7291, a mouse anti-tubulin (1/1000) and ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/1000) were also used.

The image shows increased cytoplamic staining after Pervanadate (1 mM, 30 min) treatment on Jurkat cells. The LP treatment decreased the cytoplasmic staining caused by Pervanadate.

ab40804 was used as a Pan control for ab68236. The results showed cytoplamic staining on untreated, pervanadate (1 mM, 30 min) treated and Per+LP treated Jurkat cells.

• IP

Lab

Immunoprecipitation - Anti-CD3 zeta (phospho Y83) antibody [EP776(2)Y] (AB68236)

CD3 zeta was immunoprecipitated from 0.35 mg Jurkat (Human T cell leukemia T lymphocyte) treated with pervandate (50mM 5min) whole cell lysate 10 μg with ab68236 at 1/30 dilution (2μg) . VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : Jurkat (Human T cell leukemia T lymphocyte) treated with pervandate (50mM 5min) whole cell lysate 10 μg

Lane 2 : ab68236 IP in Jurkat treated with pervandate (50mM 5min) whole cell lysate

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab68236 in Jurkat treated with pervandate (50mM 5min) whole cell lysate

Blocking and dilution buffer and concentration : 5% NFDM/TBST.

All lanes:

Immunoprecipitation - Anti-CD3 zeta (phospho Y83) antibody [EP776(2)Y] (ab68236)

Predicted band size: 19 kDa

Observed band size: 18-22 kDa

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• WB

Lab

Western blot - Anti-CD3 zeta (phospho Y83) antibody [EP776(2)Y] (AB68236)

Blocking buffer 5% NFDM/TBST

Diluting buffer 5% NFDM/TBST

All lanes:

Western blot - Anti-CD3 zeta (phospho Y83) antibody [EP776(2)Y] (ab68236) at 1/2000 dilution

Lane 1:

Untreated Jurkat cells whole cell lysates at 10 µg

Lane 2:

Jurkat cells were treated with 50mM Pervanadate for 5 minutes whole cell lysates at 10 µg

Lane 3:

Jurkat cells were treated with 50mM Pervanadate for 5 minutes whole cell lysates, then the membrane was incubated with Alkaline phosphatase at 10 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: 18 kDa

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Exposure time: 3min

• WB

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Western blot - Anti-CD3 zeta (phospho Y83) antibody [EP776(2)Y] (AB68236)

All lanes:

Western blot - Anti-CD3 zeta (phospho Y83) antibody [EP776(2)Y] (ab68236) at 1/10000 dilution

Lane 1:

Jurkat cell lysate, untreated. at 10 µg

Lane 2:

Jurkat cell lysate, treated with pervanadate at 10 µg

Secondary

All lanes:

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

Predicted band size: 19 kDa

Observed band size: 18-22 kDa

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• Dot

Unknown

Dot Blot - Anti-CD3 zeta (phospho Y83) antibody [EP776(2)Y] (AB68236)

Dot blot analysis of CD3 zeta (pY83) phospho peptide (lane 1) and CD3 zeta non-phospho peptide (lane 2) labelling CD3 zeta (phospho Y83) with ab68236 at a dilution of 1/1000. A peroxidase-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/2500).

Blocking and dilution buffer : 5% NFDM/TBST.

Exposure time : 3 minutes.