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7770
HTScan® PDGF Receptor β Kinase Assay Kit

HTScan® PDGF Receptor β Kinase Assay Kit #7770

This product is discontinued

Kinase Assay - Radiometric Image 1

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.

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Kinase Assay - DELFIA Image 2

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.)

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Kinase Assay - DELFIA Image 3

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.)

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Kinase Assay - DELFIA Image 4

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.)

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Kinase Assay - DELFIA Image 5

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.)

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Product Includes Quantity (with Count) Solution Color
DTT (1000X) 1 x 80 µl
ATP (10 mM) 9804 1 x 1 ml

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 Formula:

Peptide substrate, Biotin-peptide: 1,945 Daltons. GST-PDGFRbeta Kinase: 88 kDa.

Peptide Core Sequence:

NEY*FY*V

Platelet derived growth factor (PDGF) family proteins 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). PDGFRα homodimers bind all PDGF isoforms except those containing PDGF D. PDGFRβ homodimers bind PDGF BB and DD isoforms, as well as the PDGF AB heterodimer. The heteromeric PDGF receptor α/β 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).

  1. Deuel, T.F. et al. (1988) Biofactors 1, 213-7.
  2. Bergsten, E. et al. (2001) Nat. Cell Biol. 3, 512-516.
  3. Betsholtz, C. et al. (2001) Bioessays 23, 494-507.
  4. Coughlin, S.R. et al. (1988) Prog. Clin. Biol. Res. 266, 39-45.
  5. Ostman, A. and Heldin, C.H. (2001) Adv Cancer Res 80, 1-38.
  6. Panayotou, G. et al. (1992) EMBO J. 11, 4261-4272.
  7. Ramalingam, K. et al. (1995) Bioorg Med Chem 3, 1263-72.
  8. Kashishian, A. et al. (1992) EMBO J. 11, 1373-1382.
Entrez-Gene Id
5159
Swiss-Prot Acc.
P09619
For Research Use Only. Not For Use In Diagnostic Procedures.

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
DELFIA is a registered trademark of PerkinElmer, Inc.

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