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Analysis of LAT (pY226) in activated human T leukemia cells. Jurkat cells (ATCC TIB152) were serum starved overnight and then either stimulated with 5 mM hydrogen peroxide for 15 minutes (shaded histogram) or unstimulated (open histogram). The cells were fixed (BD Phosflow™ Fix Buffer I, Cat. No. 557870) for 10 minutes at 37°C, then permeabilized (BD Phosflow™ Perm Buffer III, Cat. No. 558050) on ice for at least 30 minutes, and then stained with PE anti-LAT (pY226, Cat. No. 558433). Flow cytometry was performed on a BD FACSCalibur™ flow cytometry system.
BD™ Phosflow PE Mouse anti-LAT (pY226)
Regulatory Status Legend
Any use of products other than the permitted use without the express written authorization of Becton, Dickinson and Company is strictly prohibited.
Preparation And Storage
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).
- Source of all serum proteins is from USDA inspected abattoirs located in the United States.
- 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.
- For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
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 J96-1238.58.93 monoclonal antibody recognizes the phosphorylated tyrosine 226 (pY226) of LAT, which is one of the phosphotyrosine sites required for binding Vav, Grb2, and Gads.
Development References (5)
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Janssen E, Zhang W. Adaptor proteins in lymphocyte activation. Curr Opin Immunol. 2003; 15:269-276. (Biology). View Reference
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Lin J, Weiss A. Identification of the minimal tyrosine residues required for linker for activation of T cell function. J Biol Chem. 2001; 276:29588-29595. (Biology). View Reference
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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). View Reference
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Samelson LE. Signal transduction mediated by the T cell antigen receptor: The role of adapter proteins. Annu Rev Immunol. 2002; 20:371-394. (Biology).
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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 Reference
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