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Alexa Fluor® 488 Mouse Anti-ZAP70 (pY319)/Syk (Y352)
Alexa Fluor® 488 Mouse Anti-ZAP70 (pY319)/Syk (Y352)

Flow cytometric analysis of Phospho-ZAP70. Jurkat cells (ATCC TIB-152) were starved overnight in RPMI containing 0.1% FCS. The following day, cells were either left untreated (unshaded) or treated (shaded) with H2O2 (5 mM for 15 minutes at 37°C). Cells were fixed in 2% paraformaldehyde (10 minutes at 37°C) and permeabilized by adding 90% methanol to the cell pellet (30 minutes on ice or overnight at -20°C). Cells were then washed twice in BD Pharmingen™ Stain Buffer (Cat. No. 554656), and stained with Phospho-ZAP70-Alexa488 (clone 17A/P-ZAP70). The cells were analyzed on a BD FACSCalibur™ flow cytometry system.

Flow cytometric analysis of Phospho-ZAP70. Jurkat cells (ATCC TIB-152) were starved overnight in RPMI containing 0.1% FCS. The following day, cells were either left untreated (unshaded) or treated (shaded) with H2O2 (5 mM for 15 minutes at 37°C). Cells were fixed in 2% paraformaldehyde (10 minutes at 37°C) and permeabilized by adding 90% methanol to the cell pellet (30 minutes on ice or overnight at -20°C). Cells were then washed twice in BD Pharmingen™ Stain Buffer (Cat. No. 554656), and stained with Phospho-ZAP70-Alexa488 (clone 17A/P-ZAP70). The cells were analyzed on a BD FACSCalibur™ flow cytometry system.

Product Details
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BD Phosflow™
ZAP-70; SRK; STD; TZK; Zeta-chain associated protein kinase, 70kD
Human (QC Testing), Mouse, Rat (Tested in Development)
Mouse IgG1
Human phosphorylated ZAP70 Peptide
Intracellular staining (flow cytometry) (Routinely Tested)
70 kDa
20 µl
AB_396885
Aqueous buffered solution containing BSA and ≤0.09% sodium azide.
RUO


Preparation And Storage

Store undiluted at 4°C and protected from prolonged exposure to light. Do not freeze. The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography. The antibody was conjugated to Alexa Fluor® 488 under optimum conditions, and unreacted Alexa Fluor® 488 was removed.

Recommended Assay Procedures

Jurkat cells treated with H2O2 are suggested as a positive control. However, other cell types or methods may also be used for detection of phosphorylated ZAP70.

Product Notices

  1. This reagent has been pre-diluted for use at the recommended Volume per Test. We typically use 1 × 10^6 cells in a 100-µl experimental sample (a test).
  2. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
  3. The Alexa Fluor®, Pacific Blue™, and Cascade Blue® dye antibody conjugates in this product are sold under license from Molecular Probes, Inc. for research use only, excluding use in combination with microarrays, or as analyte specific reagents. The Alexa Fluor® dyes (except for Alexa Fluor® 430), Pacific Blue™ dye, and Cascade Blue® dye are covered by pending and issued patents.
  4. Alexa Fluor® 488 fluorochrome emission is collected at the same instrument settings as for fluorescein isothiocyanate (FITC).
  5. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
  6. Caution: Sodium azide yields highly toxic hydrazoic acid under acidic conditions. Dilute azide compounds in running water before discarding to avoid accumulation of potentially explosive deposits in plumbing.
  7. Source of all serum proteins is from USDA inspected abattoirs located in the United States.
  8. Alexa Fluor® is a registered trademark of Molecular Probes, Inc., Eugene, OR.
  9. All other brands are trademarks of their respective owners.
557818 Rev. 2
Antibody Details
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17A/P-ZAP70

ZAP70 is a protein tyrosine kinase (PTK) that associates with the z subunit of the T cell antigen receptor (TCR) and undergoes tyrosine phosphorylation following TCR stimulation. ZAP70 contains two SH2-like domains with the PTK domain located at the C-terminus. It appears that both ZAP70 and Syk are recruited to the phosphorylated CD3 and z subunits after TCR stimulation. TCR stimulation leads to autophosphorylation of ZAP70 at Tyr-315 amd Tyr-319, and mutation of the Tyr-319 site dramatically impairs TCR signaling. In addition, TCR-mediated Lck activity leads to phosphorylation of ZAP70 on Tyr-493 in the regulatory loop of the kinase domain leading to upregulation of ZAP70 kinase activity. The significance of ZAP70 activation in mediating TCR signal transduction has been confirmed by showing that ZAP70 activity is absent in an autosomal recessive form of severe combined immunodeficiency (SCID). This is due to mutations affecting the ZAP70 kinase domain which affect the stability of the protein and TCR signaling.

Clone 17A/P-ZAP70 recognizes the phosphorylated form of ZAP70 (Y319). It also cross-reacts with SYK (Y352) due to homology of the phosphorylation site with ZAP70 (Y319). The PE-conjugated format has been evaluated using human and mouse model systems. The unconjugated form of the antibody (Cat. No. 612574) has also been shown to work in western blot analysis on human, mouse,  and rat cells.

557818 Rev. 2
Format Details
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Alexa Fluor™ 488
Alexa Fluor™ 488 Dye is part of the BD blue family of dyes. This is a small organic fluorochrome with an excitation maximum (Ex Max) at 494-nm and an emission maximum (Em Max) at 517-nm. Alexa Fluor™ 488 is designed to be excited by the Blue laser (488 nm) and detected using an optical filter centered near 520-nm (e.g., a 530/30-nm bandpass filter). Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.
Alexa Fluor™ 488
Blue 488 nm
494 nm
517 nm
557818 Rev.2
Citations & References
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Development References (3)

  1. Arpaia E, Shahar M, Dadi H, Cohen A, Roifman CM. Defective T cell receptor signaling and CD8+ thymic selection in humans lacking zap-70 kinase. Cell. 1994; 76(5):947-958. (Biology). View Reference
  2. Chan AC, Kadlecek TA, Elder ME, et al. ZAP-70 deficiency in an autosomal recessive form of severe combined immunodeficiency. Science. 1994; 264:1599-1601. (Biology).
  3. Di Bartolo V, Mege D, Germain V, et al. Tyrosine 319, a newly identified phosphorylation site of ZAP-70, plays a critical role in T cell antigen receptor signaling. J Biol Chem. 1999; 274(10):6285-6294. (Biology). View Reference
557818 Rev. 2

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Global - Refer to manufacturer's instructions for use and related User Manuals and Technical data sheets before using this products as described


Comparisons, where applicable, are made against older BD Technology, manual methods or are general performance claims.  Comparisons are not made against non-BD technologies, unless otherwise noted.

For Research Use Only. Not for use in diagnostic or therapeutic procedures.