Product Pathways - HTScan Kinase Assay Kits
HTScan® Met Kinase Assay Kit #7440
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 |
| PYK2 (Tyr402) Biotinylated Peptide # 1315 | 1.25 milliliters |
| Met Kinase # 7760 | 5 micrograms |
Description
The kit provides a means of performing kinase activity assays with recombinant human Met kinase. It includes active Met kinase (supplied as a GST fusion protein), a biotinylated peptide substrate and a phospho-tyrosine antibody for detection of the phosphorylated form of the substrate peptide.
Molecular Weights
Peptide substrate, Biotin-Pyk2 (402): 2,166 Daltons. GST-Met: 78 kDa.
Peptide Core Sequence
DIY*AE
Kinase Assay - Radiometric
Figure 1. Met 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: PolyAEKY, 1.5 µg/50 µl, recombinant Met: 50 µg/50 µl.
Kinase Assay - DELFIA
Figure 3. Dose dependence curve of Met kinase activity: DELFIA® data generated using Phospho-Tyrosine mAb (P-Tyr-100) #9411 to detect phosphorylation of substrate peptide (#1315) by Met kinase. In a 50 µl reaction, increasing amounts of Met 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 Met kinase activity: DELFIA® data generated using Phospho-Tyrosine mAb (P-Tyr-100) #9411 to detect phosphorylation of Met substrate peptide (#1315) by Met kinase. In a 50 µl reaction, 50 ng Met, 1.5 µM substrate peptide, 20 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 Met kinase activity: DELFIA® data generated using Phospho-Tyrosine mAb (P-Tyr-100) #9411 to detect phosphorylation of substrate peptide (#1315) by MET kinase. In a 50 µl reaction, 50 ng of Met 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 Met kinase activity: DELFIA® data generated using Phospho-Tyrosine mAb (P-Tyr-100) #9411 to detect phosphorylation of Met substrate peptide (#1315) by Met 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-Met Kinase fusion protein was produced using a baculovirus expression system with a construct expressing a fragment of human c-Met (Lys956-Ser1390) (GenBank accession No. X54559) with an amino-terminal GST tag. The protein was then purified by one-step affinity purification using glutathione-agarose.
Quality Control
The substrate peptide was selected using our Tyrosine Kinase Substrate Screening Kit #7450. Phospho-Tyrosine mAb (P-Tyr-100) #9411 was used for detection. The quality of the biotinylated peptide was evaluated by reverse-phase HPLC and by mass spectrometry.Purified Met kinase was quality controlled for purity by SDS-PAGE followed by coomassie stain and Western blot. The specific activity of the Met 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 Met activity using the Met substrate peptide provided in this kit. Met sensitivity to the inhibitor staurosporine was measured using the Met substrate peptide provided in this kit [Fig.5].
Background
Met, a high affinity tyrosine kinase receptor for hepatocyte growth factor (HGF, also known as scatter factor), is a disulfide-linked heterodimer made of 45 kDa α- and 145 kDa β-subunits (1,2). The α-subunit and the amino-terminal region of the β-subunit form the extracellular domain. The remainder of the β-chain spans the plasma membrane and contains a cytoplasmic region with tyrosine kinase activity. Interaction of Met with HGF results in autophosphorylation at multiple tyrosines, which recruit several downstream signaling components, including Gab1, c-Cbl and PI3 kinase (3). These fundamental events are important for all of the biological functions involving Met kinase activity. Addition of a phosphate at cytoplasmic Tyr1003 is essential for ubiquitination and Met protein degradation (4). Phosphorylation of Tyr1234/1235 in the Met kinase domain is critical to kinase activation. Phosphorylation of Tyr1349 in the Met cytoplasmic domain provides a direct binding site for Gab1 (5). Altered Met levels and/or tyrosine kinase activities are found in several types of tumors, including renal, colon and breast. Thus, Met is an attractive cancer therapeutic and diagnostic target (6).
- Weidner, K.M. et al. (1993) J. Cell Biol. 121, 145-154.
- Park, M. et al. (1986) Cell 45, 895-904.
- Bardelli, A. et al. (1997) Oncogene 15, 3103-3111.
- Taher, T.E. et al. (2002) J. Immunol. 169, 3793-3800.
- Schaeper, U. et al. (2000) J. Cell Biol. 149, 1419-1432.
- Traxler, P. et al. (2001) Med. Res. Rev. 21, 499-512.
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
- 7450 Tyrosine Kinase Substrate Screening Kit
- 7760 Met Kinase
- 9411 Phospho-Tyrosine Mouse mAb (P-Tyr-100)
- 1315 PYK2 (Tyr402) 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.
