Anti-CD3D antibody [EP4426]

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-CD3D antibody [EP4426] (AB109531)

Immunocytochemistry analysis of Jurkat (Human T cell leukemia T lymphocyte) cells labeling CD3D with Purified ab109531 at 1 : 50 dilution (2.6 μg/ml). Cells were fixed in 100% Methanol and permeabilized with 0.1% tritonX-100. Cells were counterstained with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) 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

Lab

Flow Cytometry - Anti-CD3D antibody [EP4426] (AB109531)

Flow Cytometry analysis of Jurkat (Human T cell leukemia T lymphocyte) cells labelling CD3D with Purified ab109531 at 1 : 20 dilution (5 μg/ml) (Red). Cells were fixed with 4% Paraformaldehyde and permeabilised with 90% Methanol. A Goat anti rabbit IgG (Alexa Fluor® 488, ab150081) secondary antibody was used at 1 : 2000. Isotype control - Rabbit monoclonal IgG (Black). Unlabelled control - Cell without incubation with primary antibody and secondary antibody (Blue).

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD3D antibody [EP4426] (AB109531)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human tonsil tissue sections labeling CD3D with Purified ab109531 at 1 : 100 dilution (1.3 μg/ml). Heat mediated antigen retrieval was performed using Heat mediated antigen retrieval using Bond™ Epitope Retrieval Solution 1 (pH 6.0) . Tissue was counterstained with Hematoxylin. Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) secondary antibody was used at 1 : 0 dilution. PBS instead of the primary antibody was used as the negative control.

• IP

Lab

Immunoprecipitation - Anti-CD3D antibody [EP4426] (AB109531)

Purified ab109531 at 1/50 dilution (2μg) immunoprecipitating CD3D in Jurkat whole cell lysate.
Lane 1 (input) : Jurkat (Human T cell leukemia T lymphocyte) whole cell lysate 10μg
Lane 2 (+) : ab109531 + Jurkat whole cell lysate.
Lane 3 (-) : Rabbit monoclonal IgG (ab172730) instead of ab109531 in Jurkat whole cell lysate.
VeriBlot for IP Detection Reagent (HRP) (ab131366) (1/1000 dilution) was used for Western blotting.
Blocking Buffer and concentration : 5% NFDM/TBST.
Diluting buffer and concentration : 5% NFDM/TBST.
Observed band size : 23 kDa

All lanes:

Immunoprecipitation - Anti-CD3D antibody [EP4426] (ab109531)

Predicted band size: 19 kDa

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• WB

Supplier Data

Western blot - Anti-CD3D antibody [EP4426] (AB109531)

All lanes:

Western blot - Anti-CD3D antibody [EP4426] (ab109531) at 1/1000 dilution

Lane 1:

Jurkat (Human T cell leukemia T lymphocyte) whole cell lysate at 20 µg

Lane 2:

HuT-78 (Human Sezary syndrome cutaneous T lymphocyte) whole cell lysate at 20 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

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• WB

Lab

Western blot - Anti-CD3D antibody [EP4426] (AB109531)

Lanes 1 - 6 : Merged signal (red and green). Green - ab109531 observed at 23 kDa. Red - loading control, ab8245, observed at 37 kDa.

This blot was produced using a 4-12% Bis-tris gel under the MES buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using Licor blocking buffer before being incubated with ab109531 and ab8245 (loading control) overnight at 4°C. Antibody binding was detected using Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) at a 1 : 10000 dilution for 1hr at room temperature and then imaged.

All lanes:

Western blot - Anti-CD3D antibody [EP4426] (ab109531) at 1/1000 dilution

Lane 1:

THP1 whole cell lysate (-ve control) at 15 µg

Lane 2:

Raji whole cell lysate (-ve control) at 15 µg

Lane 3:

Jurkat whole cell lysate at 15 µg

Lane 4:

Human Thymus tissue lysate at 15 µg

Lane 5:

Mouse Thymus tissue lysate at 15 µg

Lane 6:

Rat Thymus tissue lysate at 15 µg

Secondary

All lanes:

Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/10000 dilution

Predicted band size: 19 kDa

Observed band size: 23 kDa

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