PE Rabbit anti-Human/Mouse Phospho-Zap70-Y319+Syk-Y352 Monoclonal Antibody for Intracellular Flow Cytometry - P43403

Core Product Specifications and Parameters

Biological Background: ZAP70 Function and Localization

• ZAP70 is a 70 kDa tyrosine kinase essential for adaptive immunity, playing a central role in T-cell receptor (TCR) signaling by phosphorylating adapter proteins LAT and LCP2 upon activation.
• Following TCR engagement, ZAP70 is recruited to the phosphorylated ITAM motifs of CD3E and CD247/CD3Z at the plasma membrane, a process that relieves its autoinhibition and requires LCK-mediated phosphorylation (PubMed:7509083).
  Related references: PMID:7509083
• ZAP70 further regulates T-cell activation dynamics by controlling cytoskeleton reorganization, adhesion, and mobility, ensuring proper effector delivery to antigen-presenting cells.
• It is also involved in TCR downregulation by mediating CD247/CD3Z internalization and degradation through interaction with the ubiquitin ligase CBL and adaptors SLA/SLA2, thereby modulating signal termination.
• ZAP70 is expressed in T-cells, natural killer cells, early thymocytes, and pro/pre B-cells, and contributes to both T-cell and B-cell development and activation.
• The protein contains two SH2 domains that specifically bind phosphorylated ITAMs, an ATP-binding kinase domain, and undergoes post-translational modifications including phosphorylation, acetylation, and ubiquitination, as highlighted by UniProt keywords such as "Phosphoprotein", "Ubl conjugation", "Isopeptide bond", and "Acetylation".
• Alternative splicing produces multiple isoforms of ZAP70, and disease-associated variants are linked to severe combined immunodeficiency (SCID) due to impaired TCR signaling.
• The immunogen site (Y319) is a key regulatory phosphorylation site in the interdomain B region, highly conserved across human and mouse, and its phosphorylation is critical for ZAP70 activation; the antibody may also cross-react with Syk at its analogous Y352 due to sequence homology in the activation loop.

Experimental Guidance and Technical Tips

• Due to the phospho-specific nature of this antibody, include phosphatase inhibitors in all lysis, fixation, and staining buffers to maintain phosphorylation levels during sample processing.
• Validate antibody specificity by comparing signals in stimulated vs. unstimulated cells, or by treating samples with lambda phosphatase before staining.
• The antibody is validated for intracellular flow cytometry (FC intra); ensure optimized fixation and permeabilization protocols (e.g., using methanol or saponin-based permeabilization) for best results.
• For T-cell signaling studies, consider using known positive controls such as anti-CD3/CD28-stimulated Jurkat cells or primary human PBMCs, and negative controls like resting T-cells or Syk-deficient B-cells if assessing cross-reactivity.

CamelBio: Your One-Stop Sourcing Bridge

CamelBio serves as your integrated one-stop bridge for IVD raw material sourcing, covering every step from concept to clinic. For ZAP70 and Syk kinase signaling research, we provide a comprehensive portfolio of validated phospho-specific and total protein antibodies, recombinant antigens, bulk ancillary reagents, and access to rare target raw materials. Partner with us to streamline your diagnostic development with reliable, high-quality immunoassay components and technical support.