Revision 1

#7758Store at -80C

Cell Signaling Technology

Orders: 877-616-CELL (2355) [email protected]

Support: 877-678-TECH (8324)

Web: [email protected] cellsignal.com

3 Trask LaneDanversMassachusetts01923USA
For Research Use Only. Not for Use in Diagnostic Procedures.
:

:

:

:

UniProt ID:

#P06239

Entrez-Gene Id:

3932

Product Information

Storage

Antibodies are supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA and 50% glycerol. Do not aliquot the antibodies. Peptides are supplied at 6 µM in 0.001% DMSO.
Enzyme is supplied in 50 mM Tris-HCl, pH7.5; 150 mM NaCl, 0.25 mM DTT, 0.1mM EGTA, 0.1 mM EDTA, 0.1 mM PMSF, 25% glycerol, 7 mM glutathione.
Store at –80°C.
Keep enzymes on ice during use.
Avoid repeated freeze-thaw cycles.

Source / Purification

The GST-Kinase fusion protein was produced using a baculovirus expression system with a construct expressing a fragment of human Lck (Met1-Pro509) (GenBank accession No. NM_005356) with an amino-terminal GST tag. The protein was purified by one-step affinity chromatography using glutathione-agarose.

Product Description

The kit provides a means of performing enzymatic assays with active human Lck kinase. It includes active Lck kinase (supplied as a GST fusion protein), a biotinylated substrate peptide and a phospho-tyrosine monoclonal antibody for detection of the phosphorylated form of the substrate peptide.
Molecular Formula Peptide substrate, Biotin-FHIT (Tyr114): 2,150 Daltons, GST-Lck Kinase: 84 kDa.

Background

The Src family of protein tyrosine kinases, which includes Src, Lyn, Fyn, Yes, Lck, Blk, and Hck, are important in the regulation of growth and differentiation of eukaryotic cells (1). Src activity is regulated by tyrosine phosphorylation at two sites, but with opposing effects. While phosphorylation at Tyr416 in the activation loop of the kinase domain upregulates enzyme activity, phosphorylation at Tyr527 in the carboxy-terminal tail by Csk renders the enzyme less active (2).
Lck is essential for T-lymphocyte activation and differentiation (3,4). Phosphorylation of Tyr505 in the carboxy-terminal tail of Lck downregulates its catalytic activity, while phosphorylation of Tyr394 leads to an increase in Lck activity (5).

  1. Thomas, S.M. and Brugge, J.S. (1997) Annu Rev Cell Dev Biol 13, 513-609.
  2. Hunter, T. (1987) Cell 49, 1-4.
  3. Molina, T.J. et al. (1992) Nature 357, 161-164.
  4. Straus, D.B. and Weiss, A. (1992) Cell 70, 585-593.
  5. Chow, L.M. et al. (1993) Nature 365, 156-160.

Species Reactivity

Species reactivity is determined by testing in at least one approved application (e.g., western blot).

Cross-Reactivity Key

H: human M: mouse R: rat Hm: hamster Mk: monkey Vir: virus Mi: mink C: chicken Dm: D. melanogaster X: Xenopus Z: zebrafish B: bovine Dg: dog Pg: pig Sc: S. cerevisiae Ce: C. elegans Hr: horse GP: Guinea Pig Rab: rabbit All: all species expected

Trademarks and Patents

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.

Limited Uses

Except as otherwise expressly agreed in a writing signed by a legally authorized representative of CST, the following terms apply to Products provided by CST, its affiliates or its distributors. Any Customer's terms and conditions that are in addition to, or different from, those contained herein, unless separately accepted in writing by a legally authorized representative of CST, are rejected and are of no force or effect.

Products are labeled with For Research Use Only or a similar labeling statement and have not been approved, cleared, or licensed by the FDA or other regulatory foreign or domestic entity, for any purpose. Customer shall not use any Product for any diagnostic or therapeutic purpose, or otherwise in any manner that conflicts with its labeling statement. Products sold or licensed by CST are provided for Customer as the end-user and solely for research and development uses. Any use of Product for diagnostic, prophylactic or therapeutic purposes, or any purchase of Product for resale (alone or as a component) or other commercial purpose, requires a separate license from CST. Customer shall (a) not sell, license, loan, donate or otherwise transfer or make available any Product to any third party, whether alone or in combination with other materials, or use the Products to manufacture any commercial products, (b) not copy, modify, reverse engineer, decompile, disassemble or otherwise attempt to discover the underlying structure or technology of the Products, or use the Products for the purpose of developing any products or services that would compete with CST products or services, (c) not alter or remove from the Products any trademarks, trade names, logos, patent or copyright notices or markings, (d) use the Products solely in accordance with CST Product Terms of Sale and any applicable documentation, and (e) comply with any license, terms of service or similar agreement with respect to any third party products or services used by Customer in connection with the Products.

Revision 1
#7758

HTScan® Lck Kinase Assay Kit

HTScan® Lck Kinase Assay Kit: Image 1 Expand Image
Figure 3. Dose dependence curve of Lck kinase activity: DELFIA® data generated using Phospho-Tyrosine mAb (P-Tyr-100) #9411 to detect phosphorylation of substrate peptide #1365 by Lck kinase. In a 50 µl reaction, increasing amounts of Lck and 1.5 µM substrate peptide were used per reaction well at 25ºC for 30 minutes. (DELFIA® is a registered trademark of PerkinElmer, Inc.)
HTScan® Lck Kinase Assay Kit: Image 2 Expand Image
Figure 5. Staurosporine inhibition of Lck kinase activity: DELFIA® data generated using Phospho-Tyrosine mAb (P-Tyr-100) #9411 to detect phosphorylation of Lck substrate peptide (#1365) by Lck kinase. In a 50 µl reaction, 50 ng Lck, 1.5 µM substrate peptide, 50 µM ATP and increasing amounts of staurosporine were used per reaction at room temperature for 30 minutes. (DELFIA® is a registered trademark of PerkinElmer, Inc.)
HTScan® Lck Kinase Assay Kit: Image 3 Expand Image
Figure 4. Peptide concentration dependence of Lck kinase activity: DELFIA® data generated using Phospho-Tyrosine mAb (P-Tyr-100) #9411 to detect phosphorylation of substrate peptide (#1365) by Lck kinase. In a 50 µl reaction, 50 ng of Lck and increasing concentrations of substrate peptide were used per reaction at room temperature for 30 minutes. (DELFIA® is a registered trademark of PerkinElmer, Inc.)
HTScan® Lck Kinase Assay Kit: Image 4 Expand Image
Figure 2. Time course of Lck kinase activity: DELFIA® data generated using Phospho-Tyrosine mAb (P-Tyr-100) #9411 to detect phosphorylation of Lck substrate peptide (#1365) by Lck kinase. In a 50 µl reaction, 50 ng and 1.5 µM substrate peptide were used per reaction. (DELFIA® is a registered trademark of PerkinElmer, Inc.)
HTScan® Lck Kinase Assay Kit: Image 5 Expand Image
Figure 1. Lck kinase activity was measured in a radiometric assay using the following reaction conditions: 5 mM MOPS, pH 7.2, 2.5 mM β-glycerophosphate, 1 mM EGTA, 0.4 mM EDTA, 5 mM MgCl2, 0.05 mM DTT, 50 μM ATP, Substrate: Poly (EY 4:1) 400 ng/μL, and variable amount of Lck kinase.