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Alexa Fluor® 488 Mouse anti-Syk (pY348)
Alexa Fluor® 488 Mouse anti-Syk (pY348)

Flow cytometric analysis for Syk (pY348).  Jurkat cells (Human T-cell leukemia; ATCC TIB-152) were serum starved overnight, then either treated with 5 mM H2O2 at 37°C for 15 minutes (shaded histogram) or were untreated (open histogram). The cells were harvested and fixed with pre-warmed BD Cytofix™ Fixation Buffer (Cat. No. 554655) for 10 minutes at 37°C, permeabilized with BD Phosflow™ Perm buffer II (Cat. No. 558052) on ice for 30 minutes, and then stained with Alexa Fluor® 488 Mouse anti-Syk (pY348). The histograms were derived from the light scattering characteristics of the viable cells. The data demonstrates the upregulated phosphorylation of the treated cells versus untreated cells. Flow cytometry was performed on a BD FACSCalibur™ flow cytometry system.

Flow cytometric analysis for Syk (pY348).  Jurkat cells (Human T-cell leukemia; ATCC TIB-152) were serum starved overnight, then either treated with 5 mM H2O2 at 37°C for 15 minutes (shaded histogram) or were untreated (open histogram). The cells were harvested and fixed with pre-warmed BD Cytofix™ Fixation Buffer (Cat. No. 554655) for 10 minutes at 37°C, permeabilized with BD Phosflow™ Perm buffer II (Cat. No. 558052) on ice for 30 minutes, and then stained with Alexa Fluor® 488 Mouse anti-Syk (pY348). The histograms were derived from the light scattering characteristics of the viable cells. The data demonstrates the upregulated phosphorylation of the treated cells versus untreated cells. Flow cytometry was performed on a BD FACSCalibur™ flow cytometry system.

Product Details
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BD Phosflow™
Human (QC Testing), Mouse (Tested in Development)
Mouse BALB/c IgG1, κ
Phosphorylated Human Syk Peptide
Intracellular staining (flow cytometry) (Routinely Tested)
20 µl
AB_1645376
Aqueous buffered solution containing BSA, protein stabilizer, 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 lymphoid cell lines, peripheral blood mononuclear cells, and mouse splenocytes using BD Phosflow™ Fix Buffer I or Lyse/Fix Buffer.  Any of the three BD Phosflow™ permeabilization buffers may be used.

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. Alexa Fluor® is a registered trademark of Molecular Probes, Inc., Eugene, OR.
  6. Source of all serum proteins is from USDA inspected abattoirs located in the United States.
  7. 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.
  8. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
  9. Species cross-reactivity detected in product development may not have been confirmed on every format and/or application.
560081 Rev. 3
Antibody Details
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I120-722

Syk is a non-receptor protein-tyrosine kinase that is closely related to ZAP70 and plays crucial roles in the development and receptor-mediated signaling of most leukocytes and in vascular integrity.  Syk is expressed in hematopoietic cells, including B lymphocytes, immature (CD4, CD8 double-negative and double-positive) thymocytes, and myeloid cells, epithelial cell lines, and normal breast tissue.  Mature (CD4 or CD8 single-positive) thymocytes and peripheral αβ TCR-bearing T lymphocytes have very low or undetectable levels of Syk.  Syk contributes to the signal transduction process by binding to ITAMs (Immunoreceptor Tyrosine-based Activation Motifs) of immune receptors, including Igα and Igβ (CD79a and b), TCRζ, CD3ε, and FcRγ.  Upon receptor activation, Syk binds to phosphorylated ITAMs via its two N-terminal SH2 domains thereby activating Syk and causing tyrosines in the interdomain, between the SH2 and Kinase domains of Syk, to undergo auto-phosphorylation and phosphorylation by Lyn.  The tyrosine 348 phosphorylation site (pY348) in human Syk is orthologous to tyrosine 342 in mouse and rat Syk and tyrosine 315 in human ZAP70.  This phosphorylated site can act as a binding site for other signaling molecules, such as PLCγ, Vav, and Fgr.  

The I120-772 antibody is specific for human Syk (pY348) and does not cross-react with phosphorylated Zap70.  The orthologous phosphorylation site in mouse and rat Syk is Y342.

560081 Rev. 3
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
560081 Rev.3
Citations & References
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Development References (7)

  1. Abtahian F, Guerriero A, Sebzda E, et al. . Regulation of blood and lymphatic vascular separation by signaling proteins SLP-76 and Syk. Science. 2003; 299:247-251. (Biology).
  2. Coopman PJP, Do MTH, Barth M, et al. The Syk tyrosine kinase suppresses malignant growth of human breast cancer cells. Nature. 2000; 406:742-747. (Biology).
  3. Hong JJ, Yankee TM, Harrison ML, Geahlen RL. Regulation of signaling in B cells through the phosphorylation of Syk on linker region tyrosines. J Biol Chem. 2002; 277:31703-31714. (Biology).
  4. Latour S, Veillette A. Proximal protein tyrosine kinases in immunoreceptor signaling. Curr Opin Immunol. 2001; 13:299-306. (Biology).
  5. Turner M, Schweighoffer E, Colucci F, Di Santo JP, Tybulewicz VL. Tyrosine kinase SYK; essential functions for immunoreceptor signaling. Immunol Today. 2000; 21:148-154. (Biology). View Reference
  6. Vines CM, Potter JW, Xu Y, et al. Inhibition of β2 integrin receptor and Syk kinase signaling in monocytes by the Src family kinase Fgr. Immunity. 2001; 15:507-519. (Biology).
  7. Zhang J, Benestein E, Siraganian RP. Phosphorylation of Tyr342 in the linker region of Syk is critical for FceRI signaling in mast cells. Mol Cell Biol. 2002; 22:8144-8154. (Biology).
View All (7) View Less
560081 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.