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BD Phosflow™ PE Mouse Anti-Zap70 (Y319)/Syk (Y352)
Clone 17A/P-ZAP70 (RUO)
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 PE Mouse Anti-Zap70 (Y319)/Syk (Y352). 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 PE Mouse Anti-Zap70 (Y319)/Syk (Y352). The cells were analyzed on a BD FACSCalibur™ flow cytometry system.
Product Details
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_396919
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 the dye under optimum conditions and unconjugated antibody and free dye were 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.
BD® CompBeads can be used as surrogates to assess fluorescence spillover (compensation). When fluorochrome conjugated antibodies are bound to BD® CompBeads, they have spectral properties very similar to cells. However, for some fluorochromes there can be small differences in spectral emissions compared to cells, resulting in spillover values that differ when compared to biological controls. It is strongly recommended that when using a reagent for the first time, users compare the spillover on cell and BD® CompBeads to ensure that BD® CompBeads are appropriate for your specific cellular application.
Product Notices
- 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).
- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
- For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
- 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.
- Source of all serum proteins is from USDA inspected abattoirs located in the United States.
- Species cross-reactivity detected in product development may not have been confirmed on every format and/or application.
- Please refer to http://regdocs.bd.com to access safety data sheets (SDS).
557881 Rev. 3
Antibody Details
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.
557881 Rev. 3
Format Details
PE
R-Phycoerythrin (PE), is part of the BD family of Phycobiliprotein dyes. This fluorochrome is a multimeric fluorescent phycobiliprotein with excitation maximum (Ex Max) of 496 nm and 566 nm and an emission maximum (Em Max) at 576 nm. PE is designed to be excited by the Blue (488 nm), Green (532 nm) and Yellow-Green (561 nm) lasers and detected using an optical filter centered near 575 nm (e.g., a 575/26-nm bandpass filter). As PE is excited by multiple lasers, this can result in cross-laser excitation and fluorescence spillover on instruments with various combinations of Blue, Green, and Yellow-Green lasers. Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.
PE
496 nm, 566 nm
576 nm
557881 Rev.3
Citations & References
View product citations for antibody "557881" on CiteAb
Development References (3)
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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
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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).
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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
557881 Rev. 3
Please refer to Support Documents for Quality Certificates
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.