Product Pathways - HTScan Kinase Assay Kits
HTScan® Lck Kinase Assay Kit #7758
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.
| Kit Includes | Quantity |
|---|---|
| Phospho-Tyrosine Mouse mAb (P-Tyr-100) # 9411 | 30 microliters |
| HTScan® Tyrosine Kinase Buffer (4X) # 9805 | 15 milliliters |
| DTT (1000x, 1.25 M) | 80 microliters |
| ATP (10 mM) # 9804 | 1 milliliters |
| Lck Kinase # 7487 | 5 micrograms |
| FHIT (Tyr114) Biotinylated Peptide # 1365 | 1.25 milliliters |
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 Weights
Peptide substrate, Biotin-FHIT (Tyr114): 2,150 Daltons, GST-Lck Kinase: 84 kDa.
Peptide Core Sequence
SIY*EE
Kinase Assay - Radiometric
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.
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 well at 25°C for 30 minutes. (DELFIA® is a registered trademark of PerkinElmer, Inc.)
Kinase Assay - DELFIA
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.)
Kinase Assay - DELFIA
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.)
Kinase Assay - DELFIA
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.)
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.
Quality Control
FHIT (Tyr114) peptide was selected by using Tyrosine Kinase Substrate Screening Kit #7450 to screen for Lck kinase substrates. Phospho-Tyrosine mAb #9411 was used for detection. The quality of the biotinylated peptides was evaluated by reverse-phase HPLC and by mass spectrometry.Purified Lck kinase was quality controlled for purity by SDS-PAGE followed by commassie stain. The specific activity of the Lck kinase was determined using a radiometric assay [Fig.1]. Time course [Fig.2], kinase dose-dependency [Fig.3] and substrate dose-dependency [Fig.4] assays were performed to verify Lck activity using the Lck substrate peptide provided in this kit. Lck sensitivity to the inhibitor staurosporine was measured using the Lck substrate peptide provided in this kit [Fig.5].
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, 161-164.
- Straus, D.B. and Weiss, A. (1992) Cell 70, 585-593.
- Chow, L.M. et al. (1993) Nature 365, 156-160.
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
- 7487 Lck Kinase
- 7450 Tyrosine Kinase Substrate Screening Kit
- 9411 Phospho-Tyrosine Mouse mAb (P-Tyr-100)
- 1365 FHIT (Tyr114) Biotinylated Peptide
- 1462 Phospho-FHIT (Tyr114) Biotinylated Peptide
- 9953 Staurosporine
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.
