Anti-ZAP70 Purified

EXBIO

Anti-ZAP70 Purified

7

Flow cytometry: Recommended dilution for purified antibody: 1 microg/ml, intracellular staining. Recommended protocol for primary antibody conjugates using ADG Fix and Perm kit: 1) Add 50 microl peripheral blood to a 5 ml FACS tube; 2) Proceed surface staining with appropriate amount of surface antibody (20 min at room temperature); 3) Add 100 microl of Reagent A (Fixation Medium, stored and used at room temperature); 4) Incubate for 15 minutes at room temperature; 5) Add 4 ml PBS buffer and centrifuge at 300 g for 5 minutes at room temperature; 6) Remove supernatant and add to cells pellet 100 microl Reagent B (Permeabilization Medium) and 20 microl of ZAP-70-PE antibody or 10 microl of ZAP-70-FITC antibody (red blood cells are not lysed yet); 7) Vortex at low speed for 1-2 seconds (pellet must be resuspended); 8) Incubate for 15 minutes at room temperature; 9) Add to cells mixture of 1 ml Reagent B and 3 ml PBS buffer and centrifuge at 300 g for 5 minutes at room temperature; 10) Remove supernatant and resuspend cells in 100 microl PBS buffer; 11) Analyze immediately. Western blotting: Recommended dilution: 1-2 microg/ml, positive control: MOLT-4 cells.

Flow Cytometry, ImmunoPrecipitation, Western Blot, ImmunoCytoChemistry

Research Use Only

1E7.2

1 mg/ml

IgG1

The ZAP70 (zeta-associated protein of 70 kDa) tyrosine kinase was identified as a tyrosine phosphoprotein that associates with TCR zeta subunit and undergoes tyrosine phosphorylation following TCR stimulation. ZAP70 is a Syk family tyrosine kinase primarily expressed in T and NK cells that plays an essential role in signaling through the TCR. TCR-mediated activation of T cells is crucial to the immune response. In humans, ZAP70 gene mutations resulting in lower ZAP70 protein expression levels or expression of catalytically inactive ZAP70 proteins, have been identified. ZAP70 deficiency results in the absence of mature CD8+ T cells and the prevention of TCR-mediated activation of CD4+ T cells, and it can lead to severe combined immunodeficiency. In patients with chronic lymphocytic leukemia (B-CLL), ZAP70 expression on B cell was shown to be correlated with disease progression and survival. ZAP70 contains two N-terminal SH2 domains (Src homology domain 2) and a C-terminal kinase domain. During T cell activation, the binding of ZAP-70 SH2 domains to the phosphorylated zeta subunit on the activated TCR complex causes a colocalization with the Lck tyrosine kinase that phosphorylates ZAP70 on Tyr493 in the activation loop. ZAP70 autophosphorylates multiple tyrosines in the region between the SH2 domains and the kinase domain, including the binding sites for additional SH2-containing signaling proteins such as SLP76, LAT, Lck, PLCgamma1, Vav, Shc, Ras-GAP, and Abl. ZAP70-mediated activation of these downstream effectors leads to the release of intracellular calcium stores, and the transcription of interleukin-2 and other genes important for an immune response.

Human, Mouse

2°C to 8°C

12352203