Product Pathways - HTScan Kinase Assay Kits
HTScan® PDGF Receptor β Kinase Assay Kit #7770
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 |
| FLT3 (Tyr589) Biotinylated Peptide # 1305 | 1.25 milliliters |
| PDGF Receptor β Kinase # 7392 | 5 micrograms |
Description
The kit provides a means of performing kinase activity assays with recombinant human PDGFRbeta kinase. It includes active PDGFRbeta 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: 1,945 Daltons. GST-PDGFRbeta Kinase: 88 kDa.
Peptide Core Sequence
NEY*FY*V
Kinase Assay - Radiometric
Figure 1. PDGFRβ kinase activity was measured in a radiometric assay using the following reaction conditions: 4 mM MOPS, pH 7.2, 2.5 mM β-glycerophosphate, 10 mM MnCl2, 1 mM EGTA, 0.4 mM EDTA, 4 mM MgCl2, 0.05 mM DTT, 40 ng/μL BSA, 50 μM ATP, Substrate: Poly(Glu-Tyr), 400 ng/μL and recombinant PDGFRβ: variable.
Kinase Assay - DELFIA
Figure 3. Dose dependence curve of PDGFRbeta kinase activity: DELFIA® data generated using Phospho-Tyrosine Mouse mAb (P-Tyr-100) #9411 to detect phosphorylation of substrate peptide (#1305) by PDGFRbeta kinase. In a 50 µl reaction, increasing amounts of PDGFRbeta 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 5. Staurosporine inhibition of PDGFRbeta kinase activity: DELFIA® data generated using Phospho-Tyrosine Mouse mAb (P-Tyr-100) #9411 to detect phosphorylation of PDGFRbeta substrate peptide (#1305) by PDGFRbeta kinase. In a 50 µl reaction, 50 ng PDGFRbeta, 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 PDGFRbeta kinase activity: DELFIA® data generated using Phospho-Tyrosine Mouse mAb (P-Tyr-100) #9411 to detect phosphorylation of substrate peptide (#1305) by PDGFRbeta kinase. In a 50 µl reaction, 50 ng of PDGFRbeta 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 PDGFRbeta kinase activity: DELFIA® data generated using Phospho-Tyrosine Mouse mAb (P-Tyr-100) #9411 to detect phosphorylation of PDGFRbeta substrate peptide (#1305) by PDGFRbeta kinase. In a 50 µl reaction, 50 ng PDGFRbeta 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 PDGFRbeta (Gln557-Leu1106) (GenBank Accession No. NM_002609) 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 Mouse 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 PDGFRbeta kinase was quality controlled for purity by SDS-PAGE followed by Coomassie stain and Western blot. The specific activity of the PDGFRbeta 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 PDGFbeta activity using the PDGFRbeta substrate peptide provided in this kit. PDGFRbeta sensitivity to the inhibitor staurosporine was measured using the PDGFRbeta substrate peptide provided in this kit [Fig.5].
Background
The proteins of the platelet derived growth factor (PDGF) family exist as several disulphide-bonded, dimeric isoforms (PDGF AA, PDGF AB, PDGF BB, PDGF CC and PDGF DD) that bind in a specific pattern to two closely related receptor tyrosine kinases, PDGF receptor α (PDGFRα) and PDGF receptor β (PDGFRβ). PDGFRα and PDGFRβ share 75% to 85% sequence homology between their two intracellular kinase domains while the kinase insert and carboxy-terminal tail regions display a lower level (27% to 28%) of homology (1). PDGF Receptor α homodimers bind all PDGF isoforms except those containing PDGF D. PDGF Receptor β homodimers bind PDGF BB and DD isoforms, as well as the PDGF AB heterodimer. The heteromeric PDGFα/β receptor binds PDGF B, C, and D homodimers as well as the PDGF AB heterodimer (2). PDGFRα and PDGFRβ can each form heterodimers with EGFR, which is also activated by PDGF (3). Various cells differ in the total number of receptors present and in the receptor subunit composition, which may account for responsive differences among cell types to PDGF binding (4). Ligand binding induces receptor dimerization and autophosphorylation, followed by binding and activation of cytoplasmic SH2 domain-containing signal transduction molecules such as Grb2, Src, GAP, PI3 kinase, PLCγ and Nck. A number of different signaling pathways are initiated by activated PDGF receptors and lead to control of cell growth, actin reorganization, migration and differentiation (5). Tyr751 in the kinase-insert region of PDGFRβ is the docking site for PI3 kinase (6). Phosphorylated pentapeptides derived from Tyr751 of PDGFRβ (pTyr751-Val-Pro-Met-Leu) inhibit the association of the carboxy-terminal SH2 domain of the p85 subunit of PI3 kinase with PDGFRβ (7). Tyr740 is also required for PDGFRβ mediated PI3 kinase activation (8).
- Deuel, T.F. et al. (1988) Biofactors 1, 213-217.
- Bergsten, E. et al. (2001) Nat. Cell Biol. 3, 512-516.
- Betsholtz, C. et al. (2001) Bioessays 23, 494-507.
- Coughlin, S.R. et al. (1988) Prog. Clin. Biol. Res. 266, 39-45.
- Ostman, A. and Heldin, C.H. (2001) Adv. Cancer Res. 80, 1-38.
- Panayotou, G. et al. (1992) EMBO J. 11, 4261-4272.
- Ramalingam, K. et al. (1995) Bioorg. Med. Chem. 3, 1263-1272.
- Kashishian, A. et al. (1992) EMBO J. 11, 1373-1382.
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
- 7392 PDGF Receptor β Kinase
- 9411 Phospho-Tyrosine Mouse mAb (P-Tyr-100)
- 1305 FLT3 (Tyr589) Biotinylated Peptide
- 9953 Staurosporine
- 9805 HTScan® Tyrosine Kinase Buffer (4X)
- 9804 ATP (10 mM)
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.
