PRODUCT CODE: em1901-27

ZAP70 Monoclonal Antibody (EM1901-27)

Applications

  • WB

  • IHC-P

  • FC

  • ICC

REACTIVITY

  • Human

Western blot analysis of ZAP70 on Jurkat cell lysate. Proteins were transferred to a PVDF membrane and blocked with 5% BSA in PBS for 1 hour at room temperature. The primary antibody (EM1901-27, 1/500) was used in 5% BSA at room temperature for 2 hours. Goat Anti-Mouse IgG - HRP Secondary Antibody (HA1006) at 1:5,000 dilution was used for 1 hour at room temperature.
  • Western blot analysis of ZAP70 on Jurkat cell lysate. Proteins were transferred to a PVDF membrane and blocked with 5% BSA in PBS for 1 hour at room temperature. The primary antibody (EM1901-27, 1/500) was used in 5% BSA at room temperature for 2 hours. Goat Anti-Mouse IgG - HRP Secondary Antibody (HA1006) at 1:5,000 dilution was used for 1 hour at room temperature.
  • Immunohistochemical analysis of paraffin-embedded human tonsil tissue using anti-ZAP70 antibody. The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 8.0-8.4) for 20 minutes.The tissues were blocked in 5% BSA for 30 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (EM1901-27, 1/50) for 30 minutes at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
  • Flow cytometric analysis of ZAP70 was done on Jurakt cells. The cells were fixed, permeabilized and stained with the primary antibody (EM1901-27, 1/50) (red). After incubation of the primary antibody at room temperature for an hour, the cells were stained with a Alexa Fluor 488-conjugated Goat anti-Mouse IgG Secondary antibody at 1/1000 dilution for 30 minutes.Unlabelled sample was used as a control (cells without incubation with primary antibody; black).
Western blot analysis of ZAP70 on Jurkat cell lysate. Proteins were transferred to a PVDF membrane and blocked with 5% BSA in PBS for 1 hour at room temperature. The primary antibody (EM1901-27, 1/500) was used in 5% BSA at room temperature for 2 hours. Goat Anti-Mouse IgG - HRP Secondary Antibody (HA1006) at 1:5,000 dilution was used for 1 hour at room temperature.

Applications

  • WB

  • IHC-P

  • FC

  • ICC

REACTIVITY

  • Human

SPECIFICATIONS

Product Type

Mouse monoclonal primary

Product Name

ZAP70 Monoclonal Antibody (EM1901-27)

Immunogen

Recombinant protein within human zap70 aa 250-480.

Host

Mouse

Positive Control

Jurkat cell, human tonsil tissue, human colon tissue, human spleen tissue.

Conjugation

Unconjugated

Clonality

Monoclonal

Clone Number

A1B5

PROPERTIES

Form

Liquid

Storage Condition

Store at +4C after thawing. Aliquot store at -20C. Avoid repeated freeze / thaw cycles.

Storage Buffer

1*PBS (pH7.4), 0.2% BSA, 50% Glycerol. Preservative: 0.05% Sodium Azide.

Concentration

2 ug/ul

PURIFICATION

Protein G purified.

MOLECULAR WEIGHT

70 kDa

Isotype

IgG2a

APPLICATION DILUTION

  • WB

  • 1:500-1:2,000

  • IHC-P

  • 1:50-1:100

  • FC

  • 1:50-1:100

  • ICC

  • 1:50

TARGET

UNIPROT #

PROTEIN NAME

Tyrosine-protein kinase ZAP-70

GENE NAME

ZAP70

SYNONYMS

Tyrosine-protein kinase ZAP-70 70 kDa zeta-chain associated protein Syk-related tyrosine kinase ZAP70

SEQUENCE SIMILARITIES

Belongs to the protein kinase superfamily. Tyr protein kinase family. SYK/ZAP-70 subfamily.

TISSUE SPECIFICITY

Expressed in T- and natural killer cells. Also present in early thymocytes and pro/pre B-cells.

POST-TRANSLATIONAL MODIFICATION

Phosphorylated on tyrosine residues upon T-cell antigen receptor (TCR) stimulation. Phosphorylation of Tyr-315 and Tyr-319 are essential for ZAP70 positive function on T-lymphocyte activation whereas Tyr-292 has a negative regulatory role. Within the C-terminal kinase domain, Tyr-492 and Tyr-493 are phosphorylated after TCR induction, Tyr-492 playing a negative regulatory role and Tyr-493 a positive. Tyr-493 is dephosphorylated by PTN22.; Ubiquitinated in response to T cell activation. Deubiquitinated by OTUD7B.

SUBCELLULAR LOCATION

Cytoplasm. Cell membrane; Peripheral membrane protein. Note=In quiescent T-lymphocytes, it is cytoplasmic. Upon TCR activation, it is recruited at the plasma membrane by interacting with CD247/CD3Z. Colocalizes together with RHOH in the immunological synapse. RHOH is required for its proper localization to the cell membrane and cytoskeleton fractions in the thymocytes (By similarity).

FUNCTION

Tyrosine kinase that plays an essential role in regulation of the adaptive immune response. Regulates motility, adhesion and cytokine expression of mature T-cells, as well as thymocyte development. Contributes also to the development and activation of primary B-lymphocytes. When antigen presenting cells (APC) activate T-cell receptor (TCR), a serie of phosphorylations lead to the recruitment of ZAP70 to the doubly phosphorylated TCR component CD247/CD3Z through ITAM motif at the plasma membrane. This recruitment serves to localization to the stimulated TCR and to relieve its autoinhibited conformation. Release of ZAP70 active conformation is further stabilized by phosphorylation mediated by LCK. Subsequently, ZAP70 phosphorylates at least 2 essential adapter proteins: LAT and LCP2. In turn, a large number of signaling molecules are recruited and ultimately lead to lymphokine production, T-cell proliferation and differentiation. Furthermore, ZAP70 controls cytoskeleton modifications, adhesion and mobility of T-lymphocytes, thus ensuring correct delivery of effectors to the APC. ZAP70 is also required for TCR-CD247/CD3Z internalization and degradation through interaction with the E3 ubiquitin-protein ligase CBL and adapter proteins SLA and SLA2. Thus, ZAP70 regulates both T-cell activation switch on and switch off by modulating TCR expression at the T-cell surface. During thymocyte development, ZAP70 promotes survival and cell-cycle progression of developing thymocytes before positive selection (when cells are still CD4/CD8 double negative). Additionally, ZAP70-dependent signaling pathway may also contribute to primary B-cells formation and activation through B-cell receptor (BCR).