Product Pathways - HTScan Kinase Assay Kits
HTScan® EphB3 Kinase Assay Kit #7716
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 |
| Btk (Tyr223) Biotinylated Peptide # 1330 | 1.25 milliliters |
| EphB3 Kinase # 7715 | 5 micrograms |
Description
The kit provides a means of performing kinase activity assays with recombinant human EphB3 kinase. It includes active EphB3 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-peptide 2,052 Daltons. GST-EphB3 Kinase: 80 kDa.
Peptide Core Sequence
LY*DY*M
Kinase Assay - Radiometric
Figure 1. EphB3 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, 0.5 µg/50 µl, recombinant EphB3: 25 ng/50 µl.
Kinase Assay - DELFIA
Figure 3. Dose dependence curve of EphB3 kinase activity: DELFIA® data generated using Phospho-Tyrosine mAb (P-Tyr-100) #9411 to detect phosphorylation of substrate peptide (#1330) by EphB3 kinase. In a 50 µl reaction, increasing amounts of EphB3 and 1.5 µM substrate peptide 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 EphB3 kinase activity: DELFIA® data generated using Phospho-Tyrosine mAb (P-Tyr-100) #9411 to detect phosphorylation of substrate peptide (#1330) by EphB3 kinase. In a 50 µl reaction, 50 ng of EphB3 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 EphB3 kinase activity: DELFIA® data generated using Phospho-Tyrosine mAb (P-Tyr-100) #9411 to detect phosphorylation of EphB3 substrate peptide (#1330) by EphB3 kinase. In a 50 µl reaction, 50 ng EphB3 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 EphB3 (Gln585-Val998) (GenBank accession No. NM_004443) with an amino-terminal GST tag. The protein was purified by one-step affinity chromatography using glutathione-agarose.
Quality Control
The substrate peptide was selected using our Tyrosine Kinase Substrate Screening Kit #7450. Phospho-Tyrosine mAb (P-Y-100) #9411 was used for detection. The quality of the biotinylated peptide was evaluated by reverse-phase HPLC and by mass spectrometry.Purified EphB3 kinase was quality controlled for purity by SDS-PAGE followed by Coomassie stain and Western blot. The specific activity of the EphB3 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 EphB3 activity using the EphB3 substrate peptide provided in this kit.
Background
The Eph receptors are the largest known family of receptor tyrosine kinases (RTKs). They can be divided into two groups based on sequence similarity and on their preference for a subset of ligands: EphA receptors bind to a glycosylphosphatidylinositol-anchored ephrin A ligand, and EphB receptors bind to ephrin B proteins that have a transmembrane and cytoplasmic domain (1,2). Eph receptors and ligands may be involved in many diseases including cancer (3). Both ephrin A and ephrin B ligands have dual functions. As RTK ligands, the ephrins stimulate the kinase activity of the Eph receptors and activate signaling pathways in receptor-expressing cells. The ephrin extracellular domain is sufficient for this function as long as it is clustered (4). The second function of ephrins has been described as ?reverse signaling,? whereby the cytoplasmic domain becomes tyrosine phosphorylated, allowing interactions with other proteins that may activate signaling pathways in the ligand-expressing cells (5). Various stimuli can induce tyrosine phosphorylation of ephrin B, including binding to EphB receptors, activation of Src kinase and stimulation by PDGF and FGF (6). Tyrosines 324/327 have been identified as major phosphorylation sites of ephrin B1 in vivo (7).
- Wilkinson, D.G. (2000) Int. Rev. Cytol. 196, 177-244.
- Klein, R. (2001) Curr. Opin. Cell Biol. 13, 196-203.
- Dodelet, V.C. and Pasquale, E.B. (2000) Oncogene 19, 5614-5619.
- Holder, N. and Klein, R. (1999) Development 126, 2033-2044.
- Bruckner, K. et al. (1997) Science 275, 1640-1643.
- Palmer, A. et al. (2002) Mol. Cell 9, 1-20.
- Kalo, M.S. et al. (2001) J. Biol. Chem. 276, 38940-38948.
Application References
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