Product Pathways - Lymphocyte Signaling
Phospho-Zap-70 (Tyr319)/Syk (Tyr352) Antibody #2701
| Applications | Reactivity | Sensitivity | MW (kDa) | Source |
|---|---|---|---|---|
| W IP IF-IC F | H M | Endogenous | 70 Zap-70, 72 Syk | Rabbit |
Applications Key:
W=Western Blotting
IP=Immunoprecipitation
IF-IC=Immunofluorescence (Immunocytochemistry)
F=Flow Cytometry
Reactivity Key:
H=Human
M=Mouse
Species cross-reactivity is determined by Western blot.
Protocols
Specificity / Sensitivity
Phospho-Zap-70 (Tyr319)/Syk (Tyr352) Antibody detects endogenous levels of Zap-70 only when phosphorylated at Tyr319. It cross-reacts with endogenous levels of Syk when phosphorylated at Tyr352.
Source / Purification
Polyclonal antibodies are produced by immunizing rabbits with a synthetic phospho-peptide (KLH-coupled) corresponding to residues surrounding Tyr319 of human Zap-70. Antibodies are purified by protein A and peptide affinity chromatography.
Western Blotting
Western blot analysis of extracts from Jurkat cells, starved for 16 hours, and treated with 2 mM H2O2 or with calf intestinal alkaline phosphatase (CIP), using Phospho-Zap-70 (Tyr319)/Syk (Tyr352) Antibody (upper) or control Zap-70 Antibody #2702 (lower).
Western Blotting
Western blot analysis of extracts from Ramos cells, untreated or treated with anti-IgM (12 µg/ ml for 2 minutes), hydrogen peroxide (10 mM for 2 minutes) or lambda phosphatase, using Phospho-Zap-70 (Tyr319)/ Syk (Tyr352) Antibody.
Flow Cytometry
Two-color flow cytometric analysis of Jurkat cells, untreated (left) or anti-CD3 activated (right), using Phospho-Zap-70 (Tyr319)/Syk (Tyr352) Antibody and Phospho-p44/42 MAPK (Thr202/Tyr204) (E10) mAb #9106. Anti-CD3 activation increases the intensity of label with both antibodies.
Flow Cytometry
Flow cytometric analysis of Jurkat cells, untreated (blue) or CD3 treated (green), using Phospho-Zap70 (Tyr319)/Syk (Tyr352) Antibody compared to a nonspecific negative control antibody (red).
IF-IC
Immunofluorescent analysis of Jurkat cells, CD3-treated (left) or untreated (right), using Phospho-Zap-70 (Tyr319)/Syk (Tyr352) Antibody (red). Blue pseudocolor = DRAQ5™ (fluorescent DNA dye).
Background
The Syk family protein tyrosine kinase Zap-70 is expressed in T and NK cells and plays a critical role in mediating T cell activation in response to T cell receptor (TCR) engagement (1). Following TCR engagement, Zap-70 is rapidly phosphorylated on several tyrosine residues through autophosphorylation and transphosphorylation by the Src family tyrosine kinase Lck (2-6). Tyrosine phosphorylation correlates with its increased Zap-70 kinase activity and downstream signaling events. Expression of Zap-70 is correlated with disease progression and survival in patients with chronic lymphocytic leukemia (CLL) (7,8).
Phosphorylation of Tyr319 is required for the assembly of a Zap-70-containing signaling complex that leads to the activation of the PLC-gamma1-dependent and Ras-dependent signaling cascades in antigen-stimulated T cells (5,6). The orthologous Tyr352 residue in Syk is also involved in the association with PLC-gamma1 (9).
- Chu, D.H. et al. (1998) Immunol. Rev. 165, 167-180.
- Iwashima, M. et al. (1994) Science 263, 1136-1139.
- Neumeister, E.N. et al. (1995) Mol. Cell Biol. 15, 3171-3178.
- Chan, A.C. et al. (1995) EMBO J. 14, 2499-2508.
- Williams, B.L. et al. (1999) EMBO J. 18, 1832-1844.
- Di Bartolo, V. et al. (1999) J. Biol. Chem. 274, 6285-6294.
- Wiestner, A. et al. (2003) Blood 101, 4944-4951.
- Crespo, M. et al. (2003) N. Engl. J. Med. 348, 1764-1775.
- Law, C. L. et al. (1996) Mol. Cell. Biol. 16, 1305-1315.
Application References
- Guntermann, C. and Alexander, D.R. (2002) CTLA-4 suppresses proximal TCR signaling in resting human CD4(+) T cells by inhibiting ZAP-70 Tyr(319) phosphorylation: A potential role for tyrosine phosphatases. J. Immunol. 168, 4420-4429. This article references the use of Phospho-Zap-70 (Tyr319)/Syk (Tyr352) Antibody in the following applications: IP Western Blotting
- Houtman, J. C. et al. (2005) Early phosphorylation kinetics of proteins involved in proximal TCR-mediated signaling pathways. J Immunol. 175 (4), 2449-2458. This article references the use of Phospho-Zap-70 (Tyr319)/Syk (Tyr352) Antibody in the following applications: Western Blotting
- Schade, A.E. and Levine, A.D. (2002) Lipid Raft Heterogeneity in Human Peripheral Blood T Lymphoblasts: A Mechanism for Regulating the Initiation of TCR Signal Transduction. J. Immunol. 168, 2233-2239. This article references the use of Phospho-Zap-70 (Tyr319)/Syk (Tyr352) Antibody in the following applications: Western Blotting
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This product is for in vitro research use only and is not intended for use in humans or animals. This product is not intended for use as therapeutic or in diagnostic procedures.
