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Alexa Fluor® 488 Mouse anti-CD247 (pY142)
Alexa Fluor® 488 Mouse anti-CD247 (pY142)

Analysis of CD247 (pY142) 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 (Cat. No. 555329) and NA/LE Mouse anti-Human CD28 (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 2 minutes (shaded histogram) or unstimulated (open histogram).  The cells were fixed (BD Phosflow™ Fix Buffer I, Cat. No. 557870) for 10-15 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-CD247(pY142, Cat. No. 558486).  Flow cytometry was performed on a BD FACSCalibur™ flow cytometry system.

Analysis of CD247 (pY142) 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 (Cat. No. 555329) and NA/LE Mouse anti-Human CD28 (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 2 minutes (shaded histogram) or unstimulated (open histogram).  The cells were fixed (BD Phosflow™ Fix Buffer I, Cat. No. 557870) for 10-15 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-CD247(pY142, Cat. No. 558486).  Flow cytometry was performed on a BD FACSCalibur™ flow cytometry system.

Product Details
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BD Phosflow™
CD3ζ
Human (QC Testing), Mouse, Rat (Predicted)
Mouse IgG2a, κ
Phosphorylated Human CD3ζ Peptide
Intracellular staining (flow cytometry) (Routinely Tested)
20 µl
AB_647104
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.

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. Source of all serum proteins is from USDA inspected abattoirs located in the United States.
  3. 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.
  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. 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.
  7. Alexa Fluor® is a registered trademark of Molecular Probes, Inc., Eugene, OR.
  8. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
558486 Rev. 3
Antibody Details
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K25-407.69

The T cell receptor (TCR), expressed by thymocytes and T lymphocytes, is a multi-component cell-surface complex responsible for recognizing antigen in the context of MHC molecules.  The antigen-specific binding component of the TCR, Ti, is a heterodimer of the variable Ig-like subunits α and β or γ and δ.  Ti is non-covalently associated with an invariant set of molecules referred to as the CD3 polypeptides, γ, δ, ε, and ζ.  The CD3 ζ polypeptide (CD3ζ) was named CD247 at the 7th Human Leukocyte Differentiation Antigens Workshop.  CD3 appears early in thymocyte differentiation and remains expressed on all mature T lymphocytes.  After antigen recognition by the TCR, CD3ζ is the primary intracellular signal transducing subunit.  It contains three ITAMs (Immunoreceptor Tyrosine-based Activation Motifs), each of which contains a pair of tyrosine residues that are phosphorylated by Lck and Fyn and are required for signal propagation.  The molecular weight of CD3ζ is 16 kDa, and it is also observed as 32-kDa homodimers or as heterodimers with the γ chain of Fc receptors.  Upon phosphorylation, the CD3ζ monomer undergoes an apparent shift in electrophoretic mobility up to 21 kDa.  

The K25-407.69 monoclonal antibody recognizes the phosphorylated tyrosine 142 (pY142) in the third ITAM domain of human CD3ζ (CD247).

558486 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
558486 Rev.3
Citations & References
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Development References (4)

  1. Alberola-Ila J, Takaki S, Kerner JD, Perlmutter RM. Differential signaling by lymphocyte antigen receptors. Annu Rev Immunol. 1997; 15:125-154. (Biology). View Reference
  2. Ernst DN, Shih CC. CD3 complex. J Biol Regul Homeost Agents. 2000; 14(3):226-229. (Biology). View Reference
  3. Kersh EN, Shaw AS, Allen PM. Fidelity of T cell activation through multistep T cell receptor ζ phosphorylation. Science. 1998; 281:572-575. (Biology). View Reference
  4. Salomon AR, Ficarro SB, Brill LM, et al. Profiling of tyrosine phosphorylation pathways in human cells using mass spectrometry. Proc Natl Acad Sci U S A. 2003; 100(2):443-448. (Biology). View Reference
View All (4) View Less
558486 Rev. 3

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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.