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      Alexa Fluor® 647 Mouse anti-LAT (pY171)
      Alexa Fluor® 647 Mouse anti-LAT (pY171)
      Analysis of LAT (pY171) in human peripheral blood lymphocytes.  Human peripheral blood mononuclear cells 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 1-2 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 30 minutes, blocked with normal mouse immunoglobulin, and then stained with Alexa Fluor® 647 Mouse anti-LAT (pY171) and PE Mouse anti-human CD3 mAb UCHT1 (Cat. No. 555333).  The figure displays the upregulated phosphorylation of LAT in activated CD3-positive T lymphocytes.  Flow cytometry was performed on a BD FACSCalibur™ flow cytometry system.
      Alexa Fluor® 647 Mouse anti-LAT (pY171)
      Analysis of LAT (pY171) in human peripheral blood lymphocytes.  Human peripheral blood mononuclear cells 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 1-2 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 30 minutes, blocked with normal mouse immunoglobulin, and then stained with Alexa Fluor® 647 Mouse anti-LAT (pY171) and PE Mouse anti-human CD3 mAb UCHT1 (Cat. No. 555333).  The figure displays the upregulated phosphorylation of LAT in activated CD3-positive T lymphocytes.  Flow cytometry was performed on a BD FACSCalibur™ flow cytometry system.
      Product Details
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      BD Phosflow™
      Human (QC Testing)
      Mouse BALB/c IgG1, κ
      Phosphorylated Human LAT
      Intracellular staining (flow cytometry) (Routinely Tested)
      20 µl
      AB_647131
      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 antibody was conjugated to Alexa Fluor® 647 under optimum conditions, and unreacted Alexa Fluor® 647 was removed. The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography.
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      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. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
      4. 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.
      5. Alexa Fluor® 647 fluorochrome emission is collected at the same instrument settings as for allophycocyanin (APC).
      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.
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      558518 Rev. 2
      Antibody Details
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      I58-1169

      Engagement of the T cell receptor (TCR) induces signal transduction pathways that enhance gene transcription and cellular proliferation and differentiation.  TCR ligation results in the recruitment and activation of multiple protein tyrosine kinases (PTKs), including lck, fyn, and ZAP70.  Adaptor proteins, such as Grb2 and SLP-76, relay the signal to downstream effector molecules.  LAT (linker for activation of T cells) is a substrate of the activated ZAP70 and functions to bridge the activated TCR and its associated PTKs with tyrosine kinase substrates.  LAT is expressed as 36- and 38-kDa forms that result from post-translational modification, and as a 42-kDa form that results from alternative splicing.  LAT is an integral membrane protein that is phosphorylated at five tyrosine sites upon TCR ligation.  Following phosphorylation, LAT binds a number of important signaling molecules, including Grb2, Vav, PLCγ1, and the p85 subunit of PI3K.  Multiple studies have shown that functional LAT is required for T lymphocyte activation and thymocyte development.

      The I58-1169 monoclonal antibody recognizes the phosphorylated tyrosine 171 (pY171) of LAT, which is one of the phosphotyrosine sites required for binding phosphoinositide 3-kinase, Grb2, and Gads.

      558518 Rev. 2
      Format Details
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      Alexa Fluor™ 647
      Alexa Fluor™ 647 Dye is part of the BD red family of dyes. This is a small organic fluorochrome with an excitation maximum (Ex Max) at 653-nm and an emission maximum (Em Max) at 669-nm. Alexa Fluor 647 is designed to be excited by the Red laser (627-640 nm) and detected using an optical filter centered near 520-nm (e.g., a 660/20 nm bandpass filter). Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.
      Alexa Fluor™ 647
      Red 627-640 nm
      653 nm
      669 nm
      558518 Rev.2
      Citations & References
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      Development References (6)

      1. Cho S, Velikovsky CA, Swaminathan CP, Houtman JCD, Samelson LE, Mariuzza RA. Structural basis for differential recognition of tyrosine-phosphorylated sites in the linker for activation of T cells (LAT) by the adaptor Gads. EMBO J. 2004; 23:1441-1451. (Biology).
      2. Janssen E, Zhang W. Adaptor proteins in lymphocyte activation. Curr Opin Immunol. 2003; 15:269-276. (Biology). View Reference
      3. Lin J, Weiss A. Identification of the minimal tyrosine residues required for linker for activation of T cell function. J Biol Chem. 2001; 276(31):29588-29595. (Biology).
      4. Paz PE, Wang S, Clarke H, Lu X, Stokoe D, Abo A. Mapping the ZAP-70 phosphorylation sites on LAT (linker for activation of T cells) required for recruitment and activation of signalling proteins in T cells. Biochem J. 2001; 356:461-471. (Biology).
      5. Samelson LE. Signal transduction mediated by the T cell antigen receptor: The role of adapter proteins. Annu Rev Immunol. 2002; 20:371-394. (Biology).
      6. Zhu M, Janssen E, Zhang W. Minimal requirement of tyrosine residues of linker for activation of T cells in TCR signaling and thymocyte development. J Immunol. 2003; 170:325-333. (Biology).
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      558518 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.

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