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Invitrogen

Phospho-ZAP70/Syk (Tyr319, Tyr352) Monoclonal Antibody (n3kobu5), PerCP-eFluor™ 710, eBioscience™

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Product Details

46-9006-42

Applications
Tested Dilution
Publications

Flow Cytometry (Flow)

5 µL (0.03 µg)/test
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Product Specifications

Species Reactivity

Human, Mouse

Host/Isotype

Mouse / IgG2b, kappa

Recommended Isotype Control

Mouse IgG2b kappa Isotype Control (eBMG2b), PerCP-eFluor™ 710, eBioscience™

Class

Monoclonal

Type

Antibody

Clone

n3kobu5

Conjugate

PerCP-eFluor™ 710 PerCP-eFluor™ 710 PerCP-eFluor™ 710

Excitation/Emission Max

482/708 nm View spectra spectra

Form

Liquid

Concentration

5 µL/Test

Purification

Affinity chromatography

Storage buffer

PBS, pH 7.2, with BSA

Contains

0.09% sodium azide

Storage conditions

4°C, store in dark, DO NOT FREEZE!

Shipping conditions

Ambient (domestic); Wet ice (international)

RRID

AB_2573856

Product Specific Information

Description: This n3kobu5 monoclonal antibody recognizes human and mouse zeta chain-associated protein of 70 kD (also known as ZAP-70) and spleen tyrosine kinase (also known as SYK) when phosphorylated on Y319 and Y352, respectively. ZAP-70 and SYK are members of the SYK protein tyrosine kinase (PTK) family that are phosphorylated and activated by Src family PTKs. ZAP-70/SYK Y319/Y352 are located in the so-called interdomain of ZAP-70/SYK (between the N-terminal dual SH2 domains and the C-terminal kinase domain).

Phosphorylation of ZAP-70 Y319 by Lck is necessary for T cell receptor (TCR)-dependent association of ZAP-70 with Lck and phospholipase C gamma and subsequent activation of calcium-dependent and Ras signaling cascades. SYK Y352 phosphorylation by Fyn/Lyn is critical for propagation of B cell receptor (BCR) signaling and for B cell development.

Specificity of this n3kobu5 clone was determined by ELISA, flow cytometry, and western blotting.

Applications Reported: This n3kobu5 antibody has been reported for use in intracellular staining followed by flow cytometric analysis.

Applications Tested: This n3kobu5 antibody has been pre-titrated and tested by intracellular staining followed by flow cytometric analysis of stimulated normal human peripheral blood cells. This can be used at 5 µL (0.03 µg) per test. A test is defined as the amount (µg) of antibody that will stain a cell sample in a final volume of 100 µL. Cell number should be determined empirically but can range from 10^5 to 10^8 cells/test.

Staining Protocol: All protocols work well for this monoclonal antibody. Use of Protocol A: Two-step protocol: intracellular (cytoplasmic) proteins allows for the greatest flexibility for detection of surface and intracellular (cytoplasmic) proteins. Use of Protocol B: One-step protocol: intracellular (nuclear) proteins is recommended for staining of transcription factors in conjunction with surface and phosphorylated intracellular (cytoplasmic) proteins. Protocol C: Two-step protocol: Fixation/Methanol allows for the greatest discrimination of phospho-specific signaling between unstimulated and stimulated samples, but with limitations on the ability to stain specific surface proteins (refer to "Clone Performance Following Fixation/Permeabilization" located in the BestProtocols Section under the Resources tab online). All Protocols can be found in the Flow Cytometry Protocols: "Staining Intracellular Antigens for Flow Cytometry Protocol" located in the BestProtocols® Section under the Resources tab online.

PerCP-eFluor® 710 emits at 710 nm and is excited with the blue laser (488 nm); it can be used in place of PerCP-Cyanine5.5. We recommend using a 710/50 bandpass filter, however, the 695/40 bandpass filter is an acceptable alternative. Please make sure that your instrument is capable of detecting this fluorochrome.

Light sensitivity: This tandem dye is sensitive to photo-induced oxidation. Please protect this vial and stained samples from light.

Fixation: Samples can be stored in IC Fixation Buffer (Product # 00-8222) (100 µL of cell sample + 100 µL of IC Fixation Buffer) or 1-step Fix/Lyse Solution (Product # 00-5333) for up to 3 days in the dark at 4°C with minimal impact on brightness and FRET efficiency/compensation. Some generalizations regarding fluorophore performance after fixation can be made, but clone specific performance should be determined empirically.

Excitation: 488 nm; Emission: 710 nm; Laser: Blue Laser.

Filtration: 0.2 µm post-manufacturing filtered.

Target Information

ZAP70 and SYK are closely related cytoplasmic tyrosine kinases that play central roles in immune receptor signaling. ZAP70 (Zeta-chain-associated protein kinase 70; gene: ZAP70) is primarily expressed in T lymphocytes and natural killer (NK) cells, where it is recruited to the T cell receptor (TCR) complex following antigen recognition. Upon activation, ZAP70 phosphorylates downstream adaptor proteins and initiates signaling cascades that drive T cell activation, proliferation, and differentiation. SYK (Spleen Tyrosine Kinase; gene: SYK) is predominantly expressed in B cells and other hematopoietic cells and functions analogously in B cell receptor (BCR) signaling, as well as in Fc receptor and integrin-mediated pathways. Although ZAP70 and SYK share structural homology and overlapping signaling mechanisms, they exhibit distinct expression patterns and immunological roles. Dysregulation of either kinase has been implicated in autoimmune disease, immunodeficiency, and hematologic malignancies, making them important targets in immunology and therapeutic research.

For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.

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Bioinformatics

Protein Aliases: 70 kDa zeta-associated protein; 70 kDa zeta-chain associated protein; spleen tyrosine kinase; Syk kinase; Syk-related kinase; syk-related tyrosine kinase; TCR-associated protein tyrosine kinase; unnamed protein product; ZAP-70; zeta chain of T cell receptor associated protein kinase 70kDa; zeta-chain (TCR) associated protein kinase 70kDa; zeta-chain associated protein kinase, 70kD

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Gene Aliases: ADMIO2; IMD48; IMD82; mrtle; mur; p72-Syk; SRK; STCD; STD; Sykb; TZK; ZAP-70

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UniProt ID: (Human) P43405, (Mouse) P48025, (Human) P43403, (Mouse) P43404

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Entrez Gene ID: (Human) 6850, (Mouse) 20963, (Human) 7535, (Mouse) 22637

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Function(s)
phosphotyrosine binding protein kinase activity protein serine/threonine kinase activity protein tyrosine kinase activity non-membrane spanning protein tyrosine kinase activity receptor binding integrin binding protein binding ATP binding interleukin-15 receptor binding kinase activity protein kinase binding phosphatase binding Toll-like receptor binding SH2 domain binding phospholipase binding scaffold protein binding protein domain specific binding ubiquitin protein ligase binding immunoglobulin receptor binding
Process(es)
cell activation lymph vessel development positive regulation of receptor internalization stimulatory C-type lectin receptor signaling pathway adaptive immune response macrophage activation involved in immune response neutrophil activation involved in immune response leukocyte activation involved in immune response serotonin secretion by platelet cell surface pattern recognition receptor signaling pathway negative regulation of inflammatory response to antigenic stimulus regulation of transcription from RNA polymerase II promoter protein phosphorylation protein import into nucleus leukocyte cell-cell adhesion integrin-mediated signaling pathway organ morphogenesis regulation of platelet activation regulation of tumor necrosis factor-mediated signaling pathway peptidyl-tyrosine phosphorylation platelet activation neutrophil chemotaxis positive regulation of protein complex assembly receptor internalization positive regulation of type I interferon production positive regulation of interleukin-10 production positive regulation of interleukin-12 production positive regulation of interleukin-4 production positive regulation of interleukin-6 production positive regulation of interleukin-8 production positive regulation of tumor necrosis factor production regulation of superoxide anion generation positive regulation of superoxide anion generation positive regulation of cell adhesion mediated by integrin positive regulation of toll-like receptor 9 signaling pathway intracellular signal transduction Fc-epsilon receptor signaling pathway Fc-gamma receptor signaling pathway involved in phagocytosis interleukin-3-mediated signaling pathway defense response to bacterium regulation of neutrophil degranulation innate immune response positive regulation of B cell differentiation positive regulation of lymphocyte differentiation positive regulation of bone resorption blood vessel morphogenesis positive regulation of inflammatory response regulation of phagocytosis regulation of immune response B cell receptor signaling pathway leukocyte migration regulation of ERK1 and ERK2 cascade cellular response to molecule of fungal origin cellular response to lipid cellular response to low-density lipoprotein particle stimulus positive regulation of monocyte chemotactic protein-1 production regulation of arachidonic acid secretion regulation of platelet aggregation apoptotic signaling pathway beta-amyloid clearance positive regulation of cold-induced thermogenesis positive regulation of cGAS/STING signaling pathway positive regulation of TORC1 signaling cellular response to beta-amyloid cellular response to lectin autophagosome assembly positive regulation of cytokine production serotonin secretion cell surface receptor signaling pathway enzyme linked receptor protein signaling pathway G-protein coupled receptor signaling pathway negative regulation of autophagy positive regulation of autophagy peptidyl-serine phosphorylation leukotriene biosynthetic process cellular response to nutrient levels positive regulation of granulocyte macrophage colony-stimulating factor production positive regulation of interleukin-3 production collagen-activated tyrosine kinase receptor signaling pathway TORC1 signaling positive regulation of mast cell degranulation beta selection positive regulation of MAPK cascade positive regulation of JUN kinase activity positive regulation of gamma-delta T cell differentiation positive regulation of alpha-beta T cell differentiation positive regulation of alpha-beta T cell proliferation protein autophosphorylation positive regulation of peptidyl-tyrosine phosphorylation positive regulation of calcium-mediated signaling antigen receptor-mediated signaling pathway negative regulation of TORC1 signaling immune response T cell activation positive thymic T cell selection negative thymic T cell selection thymic T cell selection positive regulation of T cell differentiation T cell receptor signaling pathway T cell differentiation B cell activation T cell aggregation T cell migration
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