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Mouse Monoclonal to ZAP-70

1E7.2 (IgG1)

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Background

The ZAP-70 (zeta-associated protein of 70 kDa) tyrosine kinase was identified as a tyrosine phosphoprotein that associates with TCR zeta subunit and undergoes tyrosine phosphorylation following TCR stimulation. ZAP-70 is a Syk family tyrosine kinase primarily expressed 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, ZAP-70 gene mutations resulting in lower ZAP-70 protein expression levels or expression of catalytically inactive ZAP-70 proteins, have been identified. ZAP-70 deficiency results in the absence of mature CD8+ T cells and the prevention of TCR-mediated activation of CD4+ T cells, and it can lead to severe combined immunodeficiency.
In patients with chronic lymphocytic leukemia (B-CLL), ZAP-70 expression on B cell was shown to be correlated with disease progression and survival. ZAP-70 contains two N-terminal SH2 domains (Src homology domain 2) and a C-terminal kinase domain. During T cell activation, the binding of ZAP-70 SH2 domains to the phosphorylated zeta subunit on the activated TCR complex causes a colocalization with the Lck tyrosine kinase that phosphorylates ZAP-70 on Tyr493 in the activation loop. ZAP-70 autophosphorylates multiple tyrosines in the region between the SH2 domains and the kinase domain, including the binding sites for additional SH2-containing signaling proteins such as SLP76, LAT, Lck, PLCgamma1, Vav, Shc, Ras-GAP, and Abl. ZAP-70-mediated activation of these downstream effectors leads to the release of intracellular calcium stores, and the transcription of interleukin-2 and other genes important for an immune response.

Specificity

The mouse monoclonal antibody 1E7.2 recognizes ZAP-70, a 70 kDa protein tyrosine kinase expressed in T and NK cells.
ZAP-70 is a molecule susceptible to degradation. It is recommended to use freshly prepared cell lysates (protease inhibitors are essential) to avoid non-specific staining of degradation products.
This product is for research and in vitro experimental use only. It is not to be used for any other commercial purpose. Use of this product to produce products for sale or for therapeutic or drug discovery purposes is prohibited. In order to obtain a license to use this product for commercial purposes, contact The Regents of the Univessity of California.

Regulatory Status

Immunogen

A KLH-coupled peptide corresponding to amino acids 282-307 of human ZAP-70

Species Reactivity:

  • Human
  • Mouse

Negative Species:

Applications:

  • Flow Cytometry
    Recommended dilution for purified antibody:1 μg/ml
    Application note:intracellular staining; Fix and Perm kit from An der Grub (ADG) recommended.
    Recommended protocol for primary antibody conjugates using ADG Fix and Perm kit:
    1) Add 50 µl peripheral blood to a 5 ml FACS tube
    2) Proceed surface staining with appropriate amount of surface antibody (20 min at room temperature)
    3) Add 100 µl of Reagent A (Fixation Medium, stored and used at room temperature)
    4) Incubate for 15 minutes at room temperature
    5) Add 4 ml PBS buffer and centrifuge at 300 g for 5 minutes at room temperature
    6) Remove supernatant and add to cells pellet 100 µl Reagent B (Permeabilization Medium) and 20 µl of ZAP-70-PE antibody or 10 µl of ZAP-70-FITC antibody (red blood cells are not lysed yet)
    7) Vortex at low speed for 1-2 seconds (pellet must be resuspended)
    8) Incubate for 15 minutes at room temperature
    9) Add to cells mixture of 1 ml Reagent B and 3 ml PBS buffer and centrifuge at 300 g for 5 minutes at room temperature
    10) Remove supernatant and resuspend cells in 100 µl PBS buffer.
    11) Analyze immediately
  • Immunoprecipitation
  • Western Blotting
  • Immunocytochemistry
Usage note:
Indicated dilutions are recommended starting points for use of this product. Working concentrations should be determined by the investigator.

Product Specific References

  • *Letestu R, Rawstron A, Ghia P, Villamor N, Boeckx N, Boettcher S, Buhl AM, Duerig J, Ibbotson R, Kroeber A, Langerak A, Le Garff-Tavernier M, Mockridge I, Morilla A, Padmore R, Rassenti L, Ritgen M, Shehata M, Smolewski P, Staib P, Ticchioni M, Walker C, Ajchenbaum-Cymbalista F: Evaluation of ZAP-70 expression by flow cytometry in chronic lymphocytic leukemia: A multicentric international harmonization process. Cytometry B Clin Cytom. 2006 Jul 15;70(4):309-14. [Abstract] [Full Text]
  • *Del Principe MI, Del Poeta G, Buccisano F, Maurillo L, Venditti A, Zucchetto A, Marini R, Niscola P, Consalvo MA, Mazzone C, Ottaviani L, Panetta P, Bruno A, Bomben R, Suppo G, Degan M, Gattei V, de Fabritiis P, Cantonetti M, Lo Coco F, Del Principe D, Amadori S: Clinical significance of ZAP-70 protein expression in B-cell chronic lymphocytic leukemia. Blood. 2006 Aug 1;108(3):853-61. [Abstract] [Full Text]
  • *Preobrazhensky SN, Bahler DW: Optimization of flow cytometric measurement of ZAP-70 in chronic lymphocytic leukemia. Cytometry B Clin Cytom. 2008 Mar;74(2):118-27. [Abstract] [Full Text]
  • *Gachard N, Salviat A, Boutet C, Arnoulet C, Durrieu F, Lenormand B, Leprêtre S, Olschwang S, Jardin F, Lafage-Pochitaloff M, Penther D, Sainty D, Reminieras L, Feuillard J, Béné MC: Multicenter study of ZAP-70 expression in patients with B-cell chronic lymphocytic leukemia using an optimized flow cytometry method. Haematologica. 2008 Feb;93(2):215-23. [Abstract] [Full Text]
  • *Sargent RL, Craig FE, Swerdlow SH: Comparison of Bcl-2, CD38 and ZAP-70 Expression in Chronic Lymphocytic Leukemia. Int J Clin Exp Pathol. 2009 Jun 16;2(6):574-82. [Abstract] [Full Text]
  • *Vroblova V, Vrbacky F, Hrudkova M, Jankovicova K, Schmitzova D, Maly J, Krejsek J, Smolej L: Significant change in ZAP-70 expression during the course of chronic lymphocytic leukemia. Eur J Haematol. 2010 Jun;84(6):513-7. [Abstract]
  • *Rossi FM, Del Principe MI, Rossi D, Irno Consalvo M, Luciano F, Zucchetto A, Bulian P, Bomben R, Dal Bo M, Fangazio M, Benedetti D, Degan M, Gaidano G, Del Poeta G, Gattei V: Prognostic impact of ZAP-70 expression in chronic lymphocytic leukemia: mean fluorescence intensity T/B ratio versus percentage of positive cells. J Transl Med. 2010 Mar 8;8:23. [Abstract] [Full Text]
  • *And many other.
  • For research use only. Not for drug, diagnostic or other use.

    Example Data

    1E7-2 PE


    Fig. 1. Intracellular staining of human peripheral blood lymphocytes with anti-ZAP70 (1E7.2) PE.


    1E7-2 FITC


    Fig. 2. Intracellular staining of human peripheral blood lymphocytes with anti-ZAP70 (1E7.2) FITC.

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