Revision 1
Cell Signaling Technology

Orders: 877-616-CELL (2355) [email protected]

Support: 877-678-TECH (8324)

Web: [email protected] cellsignal.com

3 Trask LaneDanversMassachusetts01923USA
For Research Use Only. Not for Use in Diagnostic Procedures.
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UniProt ID:

#P16234

Entrez-Gene Id:

5156

Product Information

Storage

Antibodies are supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA and 50% glycerol. Do not aliquot the antibodies. Peptides are supplied at 6 µM in 0.001% DMSO.
Enzymes are supplied in 50 mM Tris-HCL (pH 8.0), 150 mM NaCl, 2 mM DTT, 15 mM reduced glutathione and 20%
glycerol. Store at –80°C.
Keep enzymes on ice during use.
Avoid repeated freeze-thaw cycles.

Source / Purification

The GST-kinase fusion protein was produced using a baculovirus expression system with a construct expressing human PDGFRalpha (Lys550-Leu1090) (GenBank Accession No. NM_006206) with an amino-terminal GST tag. The protein was purified by one-step affinity chromatography using glutathione-agarose.

Product Description

The kit provides a means of performing kinase activity assays with recombinant human PDGFRalpha kinase. It includes active PDGFRalpha kinase (supplied as a GST fusion protein), a biotinylated peptide substrate and a phospho-tyrosine monoclonal antibody for detection of the phosphorylated form of the substrate peptide.
Molecular Formula Peptide substrate, Biotin-FLT3 (Tyr589): 1945 Daltons. GST-PDGFRalpha Kinase: 76 kDa.

Background

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, 213-217.
  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, 1263-1272.
  8. Kashishian, A. et al. (1992) EMBO J. 11, 1373-1382.

Species Reactivity

Species reactivity is determined by testing in at least one approved application (e.g., western blot).

Cross-Reactivity Key

H: human M: mouse R: rat Hm: hamster Mk: monkey Vir: virus Mi: mink C: chicken Dm: D. melanogaster X: Xenopus Z: zebrafish B: bovine Dg: dog Pg: pig Sc: S. cerevisiae Ce: C. elegans Hr: horse GP: Guinea Pig Rab: rabbit All: all species expected

Trademarks and Patents

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.

使用に関する制限

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Orders: 877-616-CELL (2355)•[email protected]• Support: 877-678-TECH (8324)•[email protected]• Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.
Revision 1
#7913

HTScan® PDGF Receptor α Kinase Assay Kit

HTScan® PDGF Receptor α Kinase Assay Kit: Image 1 Expand Image
Figure 3. Dose dependence curve of PDGFRalpha kinase activity: DELFIA® data generated using Phospho-Tyrosine Mouse mAb (P-Tyr-100) #9411 to detect phosphorylation of substrate peptide (#1305) by PDGFRalpha kinase. In a 50 µl reaction, increasing amounts of PDGFRalpha and 1.5 µM substrate peptide were used per reaction at room temperature for 30 minutes. (DELFIA® is a registered trademark of PerkinElmer, Inc.)
HTScan® PDGF Receptor α Kinase Assay Kit: Image 2 Expand Image
Figure 1. PDGFRalpha kinase activity was measured in a radiometric assay using the following reaction conditions: 60 mM HEPES-NaOH, pH 7.5, 5 mM MgCl2, 5 mM MnCl2, 3 µM Na-orthovanadate, 1.2 mM DTT, 100 µM ATP, 100 µM FLT3 (Tyr589) Biotinylated Peptide (#1305) and variable amount of Recombinant PDGFRalpha. Reaction mixture incubated at room temperature for 10 minutes.
HTScan® PDGF Receptor α Kinase Assay Kit: Image 3 Expand Image
Figure 5. Staurosporine inhibition of PDGFRalpha kinase activity: DELFIA® data generated using Phospho-Tyrosine Mouse mAb (P-Tyr-100) #9411 to detect phosphorylation of PDGFRalpha substrate peptide (#1305) by PDGFRalpha kinase. In a 50 µl reaction, 100 ng PDGFRalpha, 1.5 µM substrate peptide, 5 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.)
HTScan® PDGF Receptor α Kinase Assay Kit: Image 4 Expand Image
Figure 4. Peptide concentration dependence of PDGFRalpha kinase activity: DELFIA® data generated using Phospho-Tyrosine Mouse mAb (P-Tyr-100) #9411 to detect phosphorylation of substrate peptide (#1305) by PDGFRalpha kinase. In a 50 µl reaction, 100 ng of PDGFRalpha and increasing concentrations of substrate peptide were used per reaction at room temperature for 30 minutes. (DELFIA® is a registered trademark of PerkinElmer, Inc.)
HTScan® PDGF Receptor α Kinase Assay Kit: Image 5 Expand Image
Figure 2. Time course of PDGFRalpha kinase activity: DELFIA® data generated using Phospho-Tyrosine Mouse mAb (P-Tyr-100) #9411 to detect phosphorylation of PDGFRalpha substrate peptide (#1305) by PDGFRalpha kinase. In a 50 µl reaction, 100 ng PDGFRalpha and 1.5 µM substrate peptide were used per reaction. (DELFIA® is a registered trademark of PerkinElmer, Inc.)
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Orders: 877-616-CELL (2355)•[email protected]• Support: 877-678-TECH (8324)•[email protected]• Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.