Phospho-ZAP70 (Tyr493) Antibody
Description
Antibody Characteristics and Target Biology
ZAP70 (ζ-chain-associated protein kinase 70 kDa) is a Syk-family tyrosine kinase expressed in T cells and NK cells. It binds to phosphorylated immunoreceptor tyrosine-based activation motifs (ITAMs) on TCR-CD3 complexes, becoming activated through phosphorylation at Tyr493 in its catalytic domain . This phosphorylation enhances ZAP70’s kinase activity, enabling it to phosphorylate adaptor proteins like LAT and LCP2, which propagate signals for T-cell proliferation, differentiation, and cytokine production .
Phospho-ZAP70 (Tyr493) antibodies are designed to specifically recognize this phosphorylation event. Key features include:
Research Applications
These antibodies are pivotal in studying:
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T-Cell Activation Dynamics: Detecting ZAP70 phosphorylation during TCR stimulation .
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Immune Dysregulation: Associations with ZAP70 deficiencies (severe combined immunodeficiency) and overexpression in B-cell malignancies .
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Therapeutic Targeting: Evaluating kinase inhibitors or immunomodulators affecting ZAP70 activity .
For example, Arigo Biolaboratories’ anti-ZAP70 (Tyr493) antibody (ARG51658) validates phosphorylation in WB and IHC, while BioLegend’s PE-conjugated clone (396003/396004) enables flow cytometry analysis of stimulated T cells .
Key Research Findings
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Activation Mechanism: TCR engagement triggers LCK-mediated ZAP70 recruitment and phosphorylation at Tyr493, relieving autoinhibition and enabling downstream signaling .
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Negative Regulation: Dephosphorylation by PTN22 and ubiquitination by CBL terminate ZAP70 activity, preventing hyperactivation .
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Developmental Roles: ZAP70 supports thymocyte survival and CD4+/CD8+ T-cell maturation .
Technical Considerations
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Stimulation Requirements: Phospho-Tyr493 detection often requires TCR activation (e.g., anti-CD3/CD28 antibodies or PMA/ionomycin) .
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Cell-Type Specificity: Primarily expressed in T/NK cells, but some B-cell staining may occur under high stimulation .
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Buffers: Use phospho-preserving lysis buffers and phosphatase inhibitors for optimal results .
Product Specs
Target Background
- ZAP-70 was a strong prognostic biomarker for patients with CLL PMID: 29680229
- In this article, the authors demonstrated that Natural killer cells can reduce their functional role through downregulation of Syk and Zap70 kinases. PMID: 29263215
- These results identify a tight negative feedback loop in which ZAP-70-activated p38 reciprocally phosphorylates ZAP-70 and destabilizes the signaling complex. PMID: 29440413
- we have identified a new redox-active motif which is crucial for the regulation of Zap70 stability/activity. We believe that this motif has the potential to become a novel target for the development of therapeutic tools to modulate the expression/activity of kinases. PMID: 28415650
- The expression of COBLL1, LPL, and ZAP70 corresponded to patient prognosis and to IGHV mutational status, although not absolutely. When we combined all three markers together and performed the ROC analysis, AUC increased compared to the AUC of individual gene expression. PMID: 27185377
- The authors find that ZAP-70 selects its substrates by utilizing an electrostatic mechanism that excludes substrates with positively-charged residues and favors LAT and SLP-76 phosphosites that are surrounded by negatively-charged residues. PMID: 27700984
- The data describe the critical early step of directional cell movement toward SDF-1 that ZAP-70 is recruited to the CXCR4 at the leading edge of membrane and consequently modulates lamellipodia/filopodia formation and integrin activation. PMID: 28846922
- The study identified the criteria for the design of binders that specifically address either the Syk or the Zap-70 Tandem Src Homology 2 Domains, tSH2. While Syk tSH2 has a rather broad substrate scope, ZAP-70 tSH2 required a proximal arrangement of the phosphotyrosine ligands in defined strand orientation. PMID: 28767218
- ZAP-70 signaling was impaired by cholesterol depletion, further supporting the importance of membrane organization in TCR signaling. PMID: 27384937
- We conclude that ZAP70 plays a role for the homing to and/or the survival of ALL cells in the CNS and that ZAP70 may represent a therapeutic target. Furthermore, targeting CCR7/CXCR4 may be particularly promising in treating T-ALL. PMID: 27686375
- The aim of this study was to evaluate the expression of ZAP70 changing during disease progression, the intracellular interferon gamma (IFN-gamma) and IL-4 content of T and B lymphocytes and the CLL cell subset (CD5+CD19+) in CLL patients and healthy subjects, and ZAP70 correlation with cytokine production. PMID: 26376785
- in this study, authors discovered a cycle of recruitment, activation and release for Zap70 kinases at phosphorylated T-cell antigen receptors, which turned them into a 'catalytic unit' that amplified antigenic stimuli PMID: 27869819
- The histological observations suggested that the patients represent diverse cases of NHL like mature B-cell type, mature T-cell type and high grade diffuse B-cell type NHL. The findings indicate that patients with NHL may also be analyzed for status of PAX5, CD19 and ZAP70, and their transcriptional and post-translational variants for the differential diagnosis of NHL and therapy. PMID: 27748274
- Compound heterozygous mutations in ZAP70 gene is associated with leaky severe combined immunodeficiency disorder. PMID: 28124082
- The results suggest that genetic polymorphism in the 3' UTR of ZAP-70 is associated with rheumatoid arthritis susceptibility in southern Taiwanese. PMID: 26245723
- Cellular studies with ZAP70 showed that multiple lipids bind its C-terminal SH2 domain in a spatiotemporally specific manner and thereby exert exquisite spatiotemporal control over its protein binding and signaling activities in T cells. PMID: 27052731
- Whole-exome sequencing performed on five family members revealed two affected siblings to be compound heterozygous for two unique missense mutations in the 70-kD T cell receptor zeta-chain associated protein (ZAP-70). PMID: 26783323
- The data suggest that ZO-1, along with CD38 and Zap-70, plays a role in cell cycle regulation in chronic B cell leukemia, and may be used as a prognostic marker in the disease monitoring. PMID: 26306999
- A distinct set of proteins interaction partners required for chemokine-directed T cell migration is attracted by phosphotyrosine 571 of ADAP, including ZAP70. PMID: 26246585
- Activation of innate immune receptors induces an antiapoptotic signal and proliferation in ZAP-70-positive chronic lymphocytic leukemia dependent on Syk activation. PMID: 26508782
- In all, our study demonstrates that miR-631 decreases PCa cell migration and invasion by dampening ZAP70 expression. PMID: 26620225
- The differential requirements of ZAP70 and SYK during thymic development. PMID: 26187144
- Blockade of CXCR7 suppressed MIF-mediated ERK- and zeta-chain-associated protein kinase (ZAP)-70 activation PMID: 26139098
- The kinase activity of ZAP-70 stimulates negative feedback pathways that target Lck and thereby modulate the phosphorylation patterns of the immunoreceptor tyrosine-based activation motifs of t cell receptors. PMID: 25990959
- Low ZAP-70 expression is associated with B-cell chronic lymphoid leukemia. PMID: 25743836
- we observed decreased CD3 surface expression, reduced ZAP-70 abundance and increased histone H3-acetylation in activated T lymphocytes after 5 minutes of clinorotation and a transient downregulation of CD3 and stable downregulation of IL-2R PMID: 25661802
- These findings confirm the role of PTPN22 and CD28 involved in the T cell activation pathway in the development of T1D in Tunisian families. Interestingly, ZAP70 and TCRbeta/CD3z seem to contribute to the susceptibility to the disease in our population. PMID: 25448703
- In ZAP-70(+) patients with B-cell chronic lymphocytic leukaemia, the CD4/CD8 ratio was significantly below the norm indicating an active disease process. PMID: 25804237
- ZAP-70 and CD 38 positivity were detected 25% and 36%, respectively in Chronic lymphocytic leukemia patients with concordance rate of 56%, which is higher than Western literature. PMID: 24369212
- results provide the first evidence for differential expression of CD27 among CLL prognostic groups, suggest a role for ZAP-70 dependent signaling in CD27 induction and implicate CD27 in cell-cell interactions with the lymphoid tissue microenvironment PMID: 26002513
- novel biosensor ROZA-XL displays a 3-4 times greater dynamic range than its predecessor and possesses a robust baseline FRET value for ZAP-70 PMID: 25735979
- Data indicate there was substantial decline in ZAP-70 and biomarker protein levels in Vacutainer cell processing tubes (CPTs)-isolated chronic lymphocytic leukemia (CLL) cells. PMID: 25124785
- Data indicate that the decreasing trend in the expression level of TCRzeta chain, ZAP-70 kinase and epsilon Fc Receptors FcvarepsilonRIgamma was significantly associated with disease progression. PMID: 25513989
- A specific cysteine residue in the phosphotyrosine-binding pocket of each protein (Cys39 in ZAP-70, Cys206 in Syk) that is necessary for inhibition thiol-reactive compounds, was identified. PMID: 25287889
- The corrected mean fluorescence intensity (CorrMFI) represents the most promising method currently available in a routine diagnostic setting for the assessment of ZAP-70 expression in CLL patients. PMID: 24127306
- in early stage B-CLL patients, ZAP-70 upregulation is associated with distinct patterns of activation/differentiation stage subset distribution and of cytokine expression in CD4 T lymphocytes. PMID: 24166938
- ZAP-70 CpG+223 methylation represents a superior biomarker in risk-stratification of chronic lymphocytic leukemia PMID: 24868078
- ZAP-70 enhances the migration of malignant B-cells into the supportive microenvironment found in the bone marrow mainly by enhancing signaling and migration after CXCR4 stimulation. PMID: 24312539
- LAT is a modulator of CD3zeta and ZAP-70 tyrosine phosphorylation. PMID: 24204825
- Zap70 mutation reducing protein stability demonstrates rate-limiting threshold for Zap70 protein levels exists at which signalling capacity switches from nearly intact to effectively null. PMID: 24164480
- Both drugs significantly decreased the expressions of CD5 and ZAP-70. PMID: 23686733
- A deficient lipid rafts recruitment of CD3zeta/ZAP-70/Grb2, and these proteins do not merge with GM1 within the lipid rafts. PMID: 23916875
- Data indicate that inflammatory cytokine-induced increases in chronic lymphocytic leukemia (CLL) cell adhesion to stromal cells are correlated with ZAP-70 expression and blocked by PI3K inhibitor. PMID: 23981382
- investigated, using the Sequenom MassARRAY platform, polymorphisms of sixteen single-nucleotide polymorphisms (SNPs) belonging to PTPN22, CD28, CTLA-4, and ZAP-70 genes in 76 T1D patients and 162 unrelated healthy controls PMID: 24103478
- Expression of ZAP70 in CLL cells increases expression of the NF-kappaB target genes interleukin-1beta, IL6 & IL8 upon BCR triggering. ZAP70 directly amplifies NF-kappaB signalling in CLL cells which could be an underlying mechanism for its poor prognosis. PMID: 24219331
- ZAP70 expression in regulatory T cells in allergic rhinitis: effect of immunotherapy. PMID: 23786282
- CBAP indeed can function as a novel signaling component within the ZAP70/Vav1/talin complex and plays an important role in regulating chemokine-promoted T-cell trafficking. PMID: 23620790
- Evaluated expression of CD74 in chronic lymphocytic leukemia patients. CD74 expression was significantly higher in CLL group than in controls. There was positive correlation between CD74 and ZAP70 expression. PMID: 23572149
- These results suggest the involvement of the ZAP70 and PTPN6 genes in the genetic component conferring a general susceptibility to Crohn's disease and ulcerative colitis, respectively. PMID: 23406209
- Structural basis for activation of ZAP-70 by phosphorylation of the SH2-kinase linker.Autoinhibition of ZAP-70 is fully released upon phosphorylation by Src family kinases. PMID: 23530057
Q&A
Basic Research Questions
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What is Phospho-ZAP70 (Tyr493) and why is it important in immune cell signaling?
ZAP70 (Zeta-chain-associated protein kinase 70) is a tyrosine kinase expressed exclusively in T cells and natural killer cells that plays an essential role in T cell receptor (TCR) signaling. Phosphorylation of Tyrosine 493, which is found within the activation loop of the protein's kinase domain, results in increased enzymatic activity of ZAP70 . This phosphorylation event is critical because it enables ZAP70 to subsequently phosphorylate downstream targets including the adaptor molecules LAT and LCP2, facilitating the recruitment and activation of other signaling molecules that ultimately lead to T cell proliferation, differentiation, and activation . Importantly, phosphorylation of Tyr493 within the activation loop results in enzymatic activation of ZAP70 , making it a key regulatory site for monitoring T cell activation status in research applications.
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How does ZAP70 Tyr493 phosphorylation relate to other phosphorylation sites in the protein?
ZAP70 contains multiple phosphorylation sites that regulate its function:
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Tyr493: Located in the activation loop of the kinase domain; phosphorylation results in increased enzymatic activity and is required for full catalytic activity
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Tyr492: Also positioned in the activation loop but has a negative regulatory role; phosphorylation inhibits the catalytic function of ZAP70
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Tyr319/Tyr315: Located in the SH2-kinase linker region; phosphorylation enhances ZAP70 function in mediating lymphocyte signaling and creates binding sites for other signaling molecules
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Tyr292: Has a negative regulatory role; phosphorylation terminates the transient activation of ZAP70 and attenuates lymphocyte signaling
Research has shown that inhibition of ZAP70 kinase activity led to increased phosphorylation of Tyr493, suggesting a negative feedback mechanism that regulates this site and dampens ZAP70 activation . This indicates that Tyr493 phosphorylation is regulated by complex mechanisms involving both auto-phosphorylation and phosphorylation by other kinases like Lck.
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What controls should be included when using Phospho-ZAP70 (Tyr493) antibodies?
When designing experiments with Phospho-ZAP70 (Tyr493) antibodies, include these controls:
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Positive controls: Human peripheral blood lymphocytes stimulated with anti-CD3 antibodies reliably induce ZAP70 Tyr493 phosphorylation
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Negative controls:
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Specificity controls:
For immunoblotting applications, comparing the molecular weight (approximately 70 kDa) and band pattern can provide additional validation of specificity .
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What is the relationship between ZAP70 Tyr493 phosphorylation and immune disorders?
Research has established important links between ZAP70 phosphorylation status and immune disorders:
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Immunodeficiency: ZAP70 deficiency in humans is a rare cause of combined immunodeficiency (CID) or severe CID (SCID) . Laboratory evaluation of patients shows a paucity of CD8 peripheral blood T cells and normal numbers of non-functional CD4 T cells.
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Autoimmunity: Interestingly, both loss of function and dysregulated ZAP70 activity can lead to autoimmune manifestations. Among ZAP70-deficient patients, approximately 19.4% developed autoimmune diseases including autoimmune cytopenias, nephritis, and enteropathy .
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B-cell malignancies: Higher levels of ZAP70 expression are associated with certain B-cell malignancies . In chronic lymphocytic leukemia (CLL), ZAP70 enhances B-cell receptor signaling despite absent or inefficient BCR-induced tyrosine phosphorylation at Tyr493 .
Monitoring Tyr493 phosphorylation status can therefore provide valuable insights into normal versus pathological immune signaling patterns in various disease contexts.
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Technical Considerations and Troubleshooting
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What are the optimal sample preparation protocols for detecting Phospho-ZAP70 (Tyr493) in different applications?
Optimal sample preparation varies by application type:
For Western Blotting (1:1000 dilution recommended) :
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Lyse cells in buffer containing phosphatase inhibitors (sodium orthovanadate, sodium fluoride, etc.)
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Maintain samples at 4°C throughout processing
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Use 10-20 μg of total protein per lane
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Separate on 8-10% SDS-PAGE gels
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Transfer to PVDF or nitrocellulose membranes
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Block with 5% BSA rather than milk (which contains phosphatases)
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Stimulate cells appropriately (anti-CD3 for T cells)
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Fix cells quickly with formaldehyde (2-4%)
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Permeabilize with methanol (90-100%) for at least 30 minutes at -20°C
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Wash thoroughly with phosphate buffers containing BSA
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Use 5 μL antibody per million cells or per 100 μL staining volume
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Titrate antibody concentration for optimal results
For Immunoprecipitation (1:50 dilution recommended) :
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Prepare cell lysates in non-denaturing buffer with phosphatase inhibitors
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Pre-clear lysates with Protein A/G beads
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Add 4-10 μg of antibody per 1 mg of total protein
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Incubate overnight at 4°C
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Capture with Protein A/G beads
Careful preservation of phosphorylation status through immediate fixation and inclusion of phosphatase inhibitors is critical across all applications.
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How can researchers troubleshoot weak or absent signal when using Phospho-ZAP70 (Tyr493) antibodies?
When troubleshooting weak or absent signals:
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Verify stimulation conditions: Ensure proper activation of ZAP70. For T cells, anti-CD3 stimulation should induce robust phosphorylation of Tyr493 .
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Optimize fixation timing: Phosphorylation events can be transient. Studies have shown statistically significant increases in Tyr493 phosphorylation as early as 2 minutes after TCR stimulation, remaining elevated for at least 10 minutes . Time fixation appropriately.
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Check phosphatase inhibition: Ensure complete and fresh phosphatase inhibitors in all buffers. Tyrosine phosphorylation is rapidly reversed by cellular phosphatases.
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Verify antibody storage conditions: Follow manufacturer recommendations for antibody storage (typically 2-8°C for short-term and -20°C for long-term storage) .
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Test alternative epitope retrieval methods: For IHC applications, different retrieval methods may be needed to expose the phospho-epitope.
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Consider protein dephosphorylation by PTN22: Research shows that PTN22 is required for the dephosphorylation of ZAP70 Tyrosine 493 . Cell types with high PTN22 activity may show reduced phosphorylation.
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Adjust antibody concentration: Antibody concentration may need to be increased. For flow cytometry, 5 μL per million cells is recommended, but this may need optimization .
Systematic evaluation of these factors should help resolve detection issues in most experimental settings.
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What are the key considerations for quantifying Phospho-ZAP70 (Tyr493) levels in research samples?
For accurate quantification of Phospho-ZAP70 (Tyr493):
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Normalization strategy: Always normalize phospho-specific signals to total ZAP70 protein levels to account for variations in expression. This is particularly important in comparative studies.
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Appropriate controls: Include both positive controls (stimulated T cells) and negative controls (unstimulated cells, phosphatase-treated samples) in each experiment .
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Dynamic range considerations: Phosphorylation can increase many-fold upon stimulation. Ensure detection methods have sufficient dynamic range to capture these differences without saturation.
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Statistical analysis: For flow cytometry data, compare both the percentage of positive cells and the median fluorescence intensity (MFI) of the positive population.
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Standardization: When possible, include a standardized control sample across experiments to allow for inter-experimental comparison.
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Software tools: For Western blot quantification, use appropriate software that can correct for background and normalize to loading controls.
These considerations help ensure robust and reproducible quantification of phosphorylation levels across different experimental conditions.
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