Product Pathways - Tyrosine Kinase/ Adaptors
Lck Kinase #7757
Cell Signaling Technology offers a full line of protein kinases, substrates, antibody detection reagents and HTScan® kits. Browse our "Reagents for High-Throughput Screening" product listing or contact us at drugdiscovery@cellsignal.com.
Description
Purified recombinant human Lck kinase (Met1-Pro509), supplied as a GST fusion protein.
Source / Purification
The GST-Lck Kinase fusion protein was produced using a baculovirus expression system with a construct expressing 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.
Gel Staining
Figure 1. The purity of the GST-Lck fusion protein was analyzed using SDS/PAGE followed by anti-Lck Western blot (A) or Silver stain (B).
Kinase Assay - Radiometric
Figure 2. Lck kinase activity was measured in a radiometric assay using the following reaction conditions: 60 mM HEPES-NaOH, pH 7.5, 3 mM MgCl2, 3 mM MnCl2, 3 µM Na-orthovanadate, 1.2 mM DTT, ATP (variable), 2.5 µg/50 µl PEG20,000, Substrate: PolyEY, 2 µg/50 µl and 200 ng/50 µl Recombinant Lck.
Kinase Assay - DELFIA
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 at room temperature for 30 minutes. (DELFIA® is a registered trademark of PerkinElmer, Inc.)
Quality Control
The theoretical molecular weight of the GST-Lck fusion protein is 93 kDa. The purified kinase was quality controlled for purity using SDS-PAGE followed by Silver stain and Western blot [Fig.1]. Lck kinase activity was determined using a radiometric assay [Fig.2]. A kinase dose dependency assay was performed to measure Lck activity using HTScan™ Lck Kinase Assay Kit #7758 [Fig.3].
Background
The Src family of protein tyrosine kinases (including 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. Phosphorylation of Tyr416 in the activation loop of the kinase domain by Csk upregulates enzyme activity, whereas phosphorylation of Tyr527 in the carboxy-terminal tail 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).
- Thomas, S.M. and Brugge, J.S. (1997) Annu. Rev. Cell Dev. Biol. 13, 513-609.
- Hunter, T. (1987) Cell 49, 1-4.
- Molina, T.J. et al. (1992) Nature 357, 161-4.
- Straus, D.B. and Weiss, A. (1992) Cell 70, 585-93.
- Chow, L.M. et al. (1993) Nature 365, 156-60.
Application References
Have you published research involving the use of our products? If so we'd love to hear about it. Please let us know!
Companion Products
- 7758 HTScan® Lck Kinase Assay Kit
- 1365 FHIT (Tyr114) Biotinylated Peptide
- 9411 Phospho-Tyrosine Mouse mAb (P-Tyr-100)
- 9805 HTScan® Tyrosine Kinase Buffer (4X)
- 9804 ATP (10 mM)
- 9953 Staurosporine
- 7450 Tyrosine Kinase Substrate Screening Kit
This product is for in vitro research use only and is not intended for use in humans or animals. This product is not intended for use as therapeutic or in diagnostic procedures.
