LEFT: Analysis of ZAP70 (pY292) in activated human T lymphocytes. Human whole blood was either stimulated by cross-linking of CD3 with NA/LE Mouse anti-Human CD3 mAb UCHT1 (Cat. No. 555329) on ice for 15 minutes followed by Purified Goat anti-Mouse Ig (Cat. No. 553998) on ice for 15 minutes, and then allowed to undergo phosphorylation at 37°C for 5 minutes (shaded histogram), or unstimulated (open histogram). The cells were lysed and fixed with 1X BD Phosflow™ Lyse/Fix Buffer (Cat. No. 558049) for 10-15 minutes at 37ºC, permeabilized (BD Phosflow™ Perm Buffer III, Cat. No. 558050) on ice for 30 minutes, blocked with normal mouse immunoglobulin, and then stained with Alexa Fluor® 488 Mouse anti-ZAP70 (pY292) and PerCP-Cy5.5 Mouse anti-human CD3 mAb SK7 (Cat. No. 340949). Flow cytometry was performed on a BD FACSCalibur™ flow cytometry system. For data analysis, CD3-positive and -negative lymphocytes were selected by their scatter and staining profiles. The figure displays the CD3-positive T lymphocytes. Up-regulated phosphorylation of ZAP70 (pY292) was not observed in the CD3-negative lymphocytes (data not shown). RIGHT: Analysis of ZAP70 (pY292) in activated human T leukemia cells. Jurkat cells (ATCC TIB152) were either stimulated by cross-linking of CD3 and CD28 with NA/LE Mouse anti-Human CD3 mAb UCHT1 (Cat. No. 555329) and NA/LE Mouse anti-Human CD28 mAb CD28.2 (Cat. No. 555725) on ice for 15 minutes followed by Purified Goat anti-Mouse Ig (Cat. No. 553998) on ice for 15 minutes, and then allowed to undergo phosphorylation at 37°C for 5 minutes (shaded histogram), or unstimulated (open histogram). The cells were fixed (BD Cytofix™ buffer, Cat. No. 554655) for 10 minutes at 37°C, permeabilized (BD Phosflow™ Perm Buffer III, Cat. No. 558050) on ice for at least 30 minutes, blocked with normal mouse immunoglobulin, and then stained with Alexa Fluor® 488 Mouse anti-ZAP70 (pY292). Flow cytometry was performed on a BD FACSCalibur™ flow cytometry system.
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LEFT: Analysis of ZAP70 (pY292) in activated human T lymphocytes. Human whole blood was either stimulated by cross-linking of CD3 with NA/LE Mouse anti-Human CD3 mAb UCHT1 (Cat. No. 555329) on ice for 15 minutes followed by Purified Goat anti-Mouse Ig (Cat. No. 553998) on ice for 15 minutes, and then allowed to undergo phosphorylation at 37°C for 5 minutes (shaded histogram), or unstimulated (open histogram). The cells were lysed and fixed with 1X BD Phosflow™ Lyse/Fix Buffer (Cat. No. 558049) for 10-15 minutes at 37ºC, permeabilized (BD Phosflow™ Perm Buffer III, Cat. No. 558050) on ice for 30 minutes, blocked with normal mouse immunoglobulin, and then stained with Alexa Fluor® 488 Mouse anti-ZAP70 (pY292) and PerCP-Cy5.5 Mouse anti-human CD3 mAb SK7 (Cat. No. 340949). Flow cytometry was performed on a BD FACSCalibur™ flow cytometry system. For data analysis, CD3-positive and -negative lymphocytes were selected by their scatter and staining profiles. The figure displays the CD3-positive T lymphocytes. Up-regulated phosphorylation of ZAP70 (pY292) was not observed in the CD3-negative lymphocytes (data not shown). RIGHT: Analysis of ZAP70 (pY292) in activated human T leukemia cells. Jurkat cells (ATCC TIB152) were either stimulated by cross-linking of CD3 and CD28 with NA/LE Mouse anti-Human CD3 mAb UCHT1 (Cat. No. 555329) and NA/LE Mouse anti-Human CD28 mAb CD28.2 (Cat. No. 555725) on ice for 15 minutes followed by Purified Goat anti-Mouse Ig (Cat. No. 553998) on ice for 15 minutes, and then allowed to undergo phosphorylation at 37°C for 5 minutes (shaded histogram), or unstimulated (open histogram). The cells were fixed (BD Cytofix™ buffer, Cat. No. 554655) for 10 minutes at 37°C, permeabilized (BD Phosflow™ Perm Buffer III, Cat. No. 558050) on ice for at least 30 minutes, blocked with normal mouse immunoglobulin, and then stained with Alexa Fluor® 488 Mouse anti-ZAP70 (pY292). Flow cytometry was performed on a BD FACSCalibur™ flow cytometry system.
Product Details
BD Phosflow™
ZAP-70 (pY292)
Human (QC Testing)
Mouse BALB/c IgG1, κ
Phosphorylated Human ZAP70
Intracellular staining (flow cytometry) (Routinely Tested)
20 µl
AB_647102
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
This antibody conjugate is suitable for intracellular staining of human whole blood (using BD Phosflow™ Lyse/Fix Buffer) and peripheral blood mononuclear cells (using BD Cytofix™ Fixation Buffer). Any of the three BD Phosflow™ permeabilization buffers may be used.
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).
- 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.
- Alexa Fluor® 488 fluorochrome emission is collected at the same instrument settings as for fluorescein isothiocyanate (FITC).
- 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.
- For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
- Alexa Fluor® is a registered trademark of Molecular Probes, Inc., Eugene, OR.
- All other brands are trademarks of their respective owners.
- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
Companion Products
Lyse/Fix Buffer 5X RUO
Size
250 mL
Cat No.
558049
Perm Buffer III RUO
Size
125 mL
Cat No.
558050
Fixation Buffer RUO
Size
100 mL
Cat No.
554655
Perm Buffer II RUO
Size
125 mL
Cat No.
558052
Perm/Wash Buffer I RUO
Size
125 mL
Cat No.
557885
558516 Rev. 2
Antibody Details
J34-602
The 70-kDa ζ chain-associated protein (ZAP70) is a Syk-family protein tyrosine kinase (PTK) that associates with the ζ 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. TCR-mediated Lck activity leads to phosphorylation of ZAP70 on Tyrosine 493 in the regulatory loop of the PTK domain leading to upregulation of ZAP70 kinase activity. Tyrosine 292 (Y292), in the linker region between the SH2 and PTK domains, is autophosphorylated by the activated PTK domain. By binding with c-Cbl, the phosphorylated Y292 can down-regulate TCR signaling.
The J34-602 antibody recognizes ZAP70 phosphorylated at Y292.
558516 Rev. 2
Format Details
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
558516 Rev.2
Citations & References
Development References (3)
-
Kong G, Dalton M, Wardenburg JB, Straus D, Kurosaki T, Chan AC. Distinct tyrosine phosphorylation sites in ZAP-70 mediate activation and negative regulation of antigen receptor function. Mol Cell Biol. 1996; 16:5026-5035. (Biology). View Reference
-
Magnan A, Di Bartolo V, Mura AM, et al. T cell development and T cell responses in mice with mutations affecting tyrosines 292 or 315 of the ZAP-70 protein tyrosine kinase. J Exp Med. 2001; 194:491-505. (Biology). View Reference
-
Rao N, Lupher ML Jr, Ota S, Reedquist KA, Druker BJ, Band H. The linker phosphorylation site Tyr292 mediates the negative regulatory effect of Cbl on ZAP-70 in T cells. J Immunol. 2000; 164:4616-4626. (Biology). View Reference
558516 Rev. 2
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.
