zeta-Chain (TCR) Associated Protein Kinase 70kDa (ZAP70) antibody

Antigen: zeta-Chain (TCR) Associated Protein Kinase 70kDa (ZAP70)

Synonyms: SRK, STD, TZK, ZAP-70, FLJ17670, FLJ17679, Srk, mur, 70kDa, mrtle, AI327364, MGC108902, zap70, MGC80670, MGC88962, ZAP70

Clonality: Monoclonal (ZAP-03)

Host: Mouse

Reactivity: Human

Application: Flow Cytometry (FACS), Western Blotting (WB), Immunocytochemistry (ICC)

Catalog no.: ABIN125756

Additional Information

Alternative name: ZAP70

Immunogen: Bacterially expressed fusion protein representing C-terminal part (160 amino acids) ofhuman ZAP-70 with histidine tag

Format: Purified

Isotype: IgG1  (Matching secondary antibodies)

Clone: ZAP-03

Description: The ZAP70 (zeta-associated protein of 70 kDa) tyrosine kinase was identified as a tyrosinephosphoprotein that associates with TCR zeta subunit and undergoes tyrosinephosphorylation following TCR stimulation. ZAP70 is a Syk family tyrosine kinase primarilyexpressed 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, ZAP70gene mutations resulting in lower ZAP70 protein expression levels or expression ofcatalytically inactive ZAP70 proteins, have been identified. ZAP70 deficiency results in theabsence of mature CD8+ T cells and the prevention of TCR-mediated activation of CD4+ Tcells, and it can lead to severe combined immunodeficiency.In patients with chronic lymphocytic leukemia (B-CLL), ZAP70 expression on B cell wasshown to be correlated with disease progression and survival.ZAP70 is cytosolic protein migrating at 70 kDa in SDS-PAGE. It contains two N-terminalSH2 domains (Src homology domain 2) and a C-terminal kinase domain. Crystal structure ofthe ZAP70 SH2 domains in complex with a TCR zeta subunit peptide was described. DuringT cell activation, the binding of ZAP70 SH2 domains to the phosphorylated zeta subunit onthe activated TCR complex causes a colocalization with the Lck tyrosine kinase thatphosphorylates ZAP70 on Tyr493 in the activation loop. ZAP70 autophosphorylates multipletyrosines in the region between the SH2 domains and the kinase domain, including thebinding sites for additional SH2-containing signaling proteins such as SLP76, LAT, Lck,PLCgamma1, Vav, Shc, Ras-GAP, and Abl. ZAP70-mediated activation of thesedownstream effectors leads to the release of intracellular calcium stores, and thetranscription of interleukin-2 and other genes important for an immune response.

Specificity: The antibody ZAP-03 reacts with ZAP70, a 70 kDa protein tyrosine kinase expressed in Tand NK cells.ZAP70 is a molecule susceptible to degradation. It is recommended to use freshlyprepared cell lysates (protease inhibitors are essential) to avoid non-specific staining ofdegradation products.

Application Details

Application Notes: Flow Cytometry: Recommended dilution: 2-5 µg/ml. Positive control: HPB-ALL human peripheral blood T cell leukemia cell line. Application note: Intracellular staining. - Recommended dilution: 0,5 µg/ml. Positive control: HPB-ALL human peripheral blood T cell leukemia cell line. Negative control: RAMOS human Burkitt lymphoma cell line. Sample preparation: Resuspend approx. 50 mil. cells in 1 ml cold lysis buffer (1% laurylmaltoside in 20 mMTris/Cl, 100 mM NaCl pH 8. 2, 50 mM NaF including Protease inhibitor Cocktail). Incubate 60min on ice. Centrifuge to remove cell debris. Mix lysate (1: 1) with reducing LaemmliSDS-PAGE sample buffer. Boil for 5 min. Application note: Reducing conditions. SDS-PAGE (10% separating gel).

Concentration: 1 mg/ml

Purity: > 95% (by SDS-PAGE)

Purification: Purified from hybridoma culture supernatant by protein-A affinity chromatography.

Buffer: Phosphate buffered saline (PBS) with 15 mM sodium azide, approx. pH 7.4

Storage: Store at 2-8 o C. Do not use after expiration date stamped on vial label. For long-term storage aliquot and store at -20 o C. Avoid freeze/thaw cycles.

Research Area: Signaling, Tags/Labels, Immunology, Cancer, CD Antigens, Surface Receptors of Immune Cells

Restrictions: For Research Use only

Images

Western Blotting analysis (reducing conditions) of HPB-ALL peripheral blood T cell leukemia cell line.Lane 1, 2, 3, 4: immunostaining with dilution range of anti-ZAP70 (ZAP-03, 4 ug/ml (1), 2 ug/ml (2), 1 ug/ml (3), 0.5 ug/ml (4)Lane 5: immunostaining with anti-ZAP70 comparative antibodyLane 6: immunostaining with Isotype mouse IgG1 control (PPV-06, cat. no. 11-457-C100)

Publications

Publications: Hrdinka, Dráber, Stepánek et al.: "PRR7 is a transmembrane adaptor protein expressed in activated T cells involved in regulation of T cell receptor signaling and apoptosis." in: The Journal of biological chemistry, Vol. 286, Issue 22, pp. 19617-29, 2011 (PubMed).