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PE Mouse anti-Lck
PE Mouse anti-Lck

Analysis of Lck in human peripheral blood lymphocytes.  Human whole blood was lysed and fixed with 1X BD Phosflow™ Lyse/Fix Buffer (Cat. No. 558049) for 10-15 minutes at 37ºC, then permeabilized with BD Phosflow™ Perm Buffer II (Cat. No. 558052) on ice for 30 minutes, and then stained with either PE Mouse anti-Lck (Cat. No. 558496, solid-line histogram) or PE Mouse IgG1, κ Isotype Control (Cat. No. 559320, dashed-line histogram).  Flow cytometry was performed on a BD FACSCalibur™ flow cytometry system.  The figure displays lymphocytes that were selected by their scatter profile.  

Analysis of Lck in human peripheral blood lymphocytes.  Human whole blood was lysed and fixed with 1X BD Phosflow™ Lyse/Fix Buffer (Cat. No. 558049) for 10-15 minutes at 37ºC, then permeabilized with BD Phosflow™ Perm Buffer II (Cat. No. 558052) on ice for 30 minutes, and then stained with either PE Mouse anti-Lck (Cat. No. 558496, solid-line histogram) or PE Mouse IgG1, κ Isotype Control (Cat. No. 559320, dashed-line histogram).  Flow cytometry was performed on a BD FACSCalibur™ flow cytometry system.  The figure displays lymphocytes that were selected by their scatter profile.  

Product Details
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BD Phosflow™
Human (QC Testing), Mouse (Tested in Development)
Mouse BALB/c IgG1, κ
Human N-terminal Lck
Intracellular staining (flow cytometry) (Routinely Tested)
20 µl
AB_647219
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. An isotype control should be used at the same concentration as the antibody of interest.
  3. Source of all serum proteins is from USDA inspected abattoirs located in the United States.
  4. 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.
  5. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
  6. Species cross-reactivity detected in product development may not have been confirmed on every format and/or application.
  7. Please refer to http://regdocs.bd.com to access safety data sheets (SDS).
  8. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
558496 Rev. 4
Antibody Details
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MOL 171

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.  A low level of expression has been detected in B lymphocytes, but its function in B cells is unknown.  Its expression in other leukocytes is not well defined.  Members of the Src 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 MOL 171 monoclonal antibody recognizes the 56- and 60-kDa forms of human Lck protein, regardless of phosphorylation status.  It cross reacts with mouse Lck.

558496 Rev. 4
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
558496 Rev.4
Citations & References
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Development References (7)

  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. Holdorf AD, Lee K-H, Burack WR, Allen PM, Shaw AS. Regulation of Lck activity by CD4 and CD28 in the immunological synapse. Nat Immunol. 2002; 3(3):259-264. (Biology).
  3. Johnson KG, Bromley SK, Dustin ML, Thomas ML. A supramolecular basis for CD45 tyrosine phosphatase regulation in sustained T cell activation. Proc Natl Acad Sci U S A. 2000; 97:10138-10143. (Biology).
  4. 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).
  5. Moroi Y, Koga Y, Nakamura K, Ohtsu M, Kimura G, Nomoto K. Accumulation of p60 lck in HTLV-I-transformed T cell lines detected by an anti-Lck monoclonal antibody, MOL 171. Jpn J Cancer Res. 1991; 82:909-915. (Immunogen).
  6. Nakashima I, Pu M-Y, Hamaguchi M, et al. Pathway of signal delivery to murine thymocytes triggered by co-crosslinking CD3 and Thy-1 for cellular DNA fragmentation and growth inhibition. J Immunol. 1993; 151(7):3511-3520. (Clone-specific).
  7. Veillette A, Latour S, Davidson D. Negative regulation of immunoreceptor signaling. Annu Rev Immunol. 2002; 20:669-707. (Biology).
View All (7) View Less
558496 Rev. 4

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