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Alexa Fluor® 488 Mouse Anti-LCK (pY505)
Alexa Fluor® 488 Mouse Anti-LCK (pY505)
Flow cytometric analysis of Phospho-Lck (pY505) Alexa 488. Jurkat T cells (ATCC TIB 152) were either left untreated (unshaded) or treated (shaded) with H2O2 (5 mM for 15 minutes at 37°C). Cells were fixed in BD Cytofix™ buffer (Cat. No. 554655) and permeabilized using BD Phosflow™ Perm Buffer III (Cat. No. 558050) and then stained with Alexa Fluor® 488 Mouse Anti-LCK (pY505) (1 hour at room temperature; Cat. No. 557879). The cells were analyzed on a BD FACSCalibur™ flow cytometry system.
Flow cytometric analysis of Phospho-Lck (pY505) Alexa 488. Jurkat T cells (ATCC TIB 152) were either left untreated (unshaded) or treated (shaded) with H2O2 (5 mM for 15 minutes at 37°C). Cells were fixed in BD Cytofix™ buffer (Cat. No. 554655) and permeabilized using BD Phosflow™ Perm Buffer III (Cat. No. 558050) and then stained with Alexa Fluor® 488 Mouse Anti-LCK (pY505) (1 hour at room temperature; Cat. No. 557879). The cells were analyzed on a BD FACSCalibur™ flow cytometry system.
Product Details
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BD Phosflow™
Human (QC Testing), Mouse,Rat (Tested in Development)
Mouse IgG1
Phosphorylated Human Lck Peptide
Intracellular staining (flow cytometry) (Routinely Tested)
20 µl
AB_396917
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. 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. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
  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. Source of all serum proteins is from USDA inspected abattoirs located in the United States.
  9. Species cross-reactivity detected in product development may not have been confirmed on every format and/or application.
557879 Rev. 3
Antibody Details
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4/LCK-Y505

Lck is a member of the Src family of cytoplasmic protein-tyrosine kinases (PTKs) that is normally expressed exclusively in lymphoid cells, primarily T lymphocytes and NK cells.  Members of this family have several common features:  1) unique N-terminal domains, 2) attachment to cellular membranes through a myristylated N-terminus, and 3) homologous SH2, SH3, and catalytic domains.  The unique N-terminal domain of Lck interacts with the cytoplasmic tails of the CD4 and CD8 cell-surface glycoproteins of T lymphocytes, which recognize antigen presenting cells via their surface MHC class II and class I molecules, respectively.  The catalytic activity of Lck is regulated by both kinases and phosphatases that control the phosphorylation states of two tyrosine residues that have opposing effects.  Repression of Lck's catalytic activity occurs via phosphorylation at tyrosine 505 (Y505), located near the carboxy terminus.  Phosphorylation of this tyrosine site is mediated by the Csk family of PTKs, and its dephosphorylation is mediated by the protein tyrosine phosphatase, CD45.  When Lck is phosphorylated at this site, it assumes a folded tertiary structure which is enzymatically inactive.  When CD45 dephosphorylates it at Y505, Lck is able to autophosphorylate its Y394, which leads to conformational changes in the catalytic domain that induce kinase activity.  However, it has been observed that the inhibitory effect of the phosphorylated Y505 can be overcome by direct engagement of Lck's SH3 domain and that both Y394 and Y505 are phosphorylated together in cells activated by hydrogen peroxide.  Activated Lck phosphorylates the ITAMs (Immunoreceptor-based Tyrosine Activation Motifs) of the T cell receptor (TCR) and thus is critical for activation and development of T lymphocytes.  The interactions of Lck, Csk, CD45, CD4 or CD8, and TCR are only a small part of a complex immunoregulatory cascade that involves additional substrates for Csk and CD45, other enzymes, adhesion molecules, adaptor proteins, and specialized membrane microdomains.

The 4/LCK-Y505 monoclonal antibody recognizes the phosphorylated Y505 of the catalytic domain of Lck. The Alexa Fluor® 488- conjugated format has been evaluated by flow using a human model system. However, the unconjugated form of this antibody (Cat. No. 612390) has been shown to react with human, mouse, and rat in western blot. A phosphorylated peptide corresponding to residues around Tyrosine-505 from human Lck was used as the immunogen.

557879 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
557879 Rev.3
Citations & References
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Development References (3)

  1. Hardwick JS, Sefton BM. The activated form of the Lck tyrosine protein kinase in cells exposed to hydrogen peroxide is phosphorylated at both Tyr-394 and Tyr-505. J Biol Chem. 1997; 272:25429-25432. (Biology).
  2. Lee-Fruman KK, Collins TL, Burakoff SJ. Role of the Lck Src homology 2 and 3 domains in protein tyrosine phosphorylation. J Biol Chem. 1996; 271:25003-25010. (Biology).
  3. Wang, B, Lemay S, Tsai S, Veillette A. SH2 domain-mediated interaction of inhibitory protein tyrosine kinase Csk with protein tyrosine phosphatase-HSCF. Mol Cell Biol. 2001; 21:1077-1088. (Biology).
557879 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.