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PE Mouse anti-LAT (pY171)
PE 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 PE Mouse anti-LAT (pY171, Cat. No. 558487) and APC Mouse anti-human CD3 mAb UCHT1 (Cat. No. 555335).  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.

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 PE Mouse anti-LAT (pY171, Cat. No. 558487) and APC Mouse anti-human CD3 mAb UCHT1 (Cat. No. 555335).  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_647217
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 with R-PE under optimum conditions, and unconjugated antibody and free PE were 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. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
  5. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
558487 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.

558487 Rev. 2
Format Details
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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.
altImg
PE
Yellow-Green 488 nm, 532 nm, 561 nm
496 nm, 566 nm
576 nm
558487 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).
View All (6) View Less
558487 Rev. 2

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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.