The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use at an assay dependent concentration.
1/100 - 1/250.
1/5000 - 1/10000. Detects a band of approximately 18-22 kDa (predicted molecular weight: 18 kDa).
Is unsuitable for IHC-P.
Probable role in assembly and expression of the TCR complex as well as signal transduction upon antigen triggering.
Involvement in disease
Defects in CD247 are the cause of immunodeficiency due to defect in CD3-zeta (CD3ZID) [MIM:610163]. An immunological deficiency characterized by T-cells impaired immune response to alloantigens, tetanus toxoid and mitogens.
Belongs to the CD3Z/FCER1G family. Contains 3 ITAM domains.
The ITAM domains mediate interaction with SHB.
Phosphorylated on Tyr residues after T-cell receptor triggering.
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.
Western blot - Anti-CD3 zeta (phospho Y83) antibody [EP776(2)Y] (ab68236)
All lanes : Anti-CD3 zeta (phospho Y83) antibody [EP776(2)Y] (ab68236) at 1/2000 dilution
Lane 1 : Untreated Jurkat cells whole cell lysates Lane 2 : Jurkat cells were treated with 50mM Pervanadate for 5 minutes whole cell lysates Lane 3 : Jurkat cells were treated with 50mM Pervanadate for 5 minutes whole cell lysates. Then the membrane was incubated with Alkaline phosphatase.
Lysates/proteins at 10 µg per lane.
Secondary Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution
Predicted band size : 18 kDa Observed band size : 18 kDa
Exposure time : 3 minutes
Blocking buffer 5% NFDM/TBST
Diluting buffer 5% NFDM/TBST
Western blot - CD3 zeta (phospho Y83) antibody [EP776(2)Y] (ab68236)
All lanes : Anti-CD3 zeta (phospho Y83) antibody [EP776(2)Y] (ab68236) at 1/10000 dilution
Lane 1 : Jurkat cell lysate, untreated. Lane 2 : Jurkat cell lysate, treated with pervanadate
Lysates/proteins at 10 µg per lane.
Secondary HRP-conjugated goat anti-rabbit IgG at 1/2000 dilution
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.
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).