Figure 4. PDGFR alpha kinase activity was measured in a radioisotopic filtration assay using the following reaction conditions: 60 mM HEPES-NaOH, pH 7.5, 3 mM MgCl2, 3 mM MnCl2, 3 uM Na-orthovanadate, 1.2 mM DTT, ATP (variable), 2.5 ug/50 ul PEG20.000, Substrate: Poly(AEKY) 2.5 ug/50 ul, Recombinant PDGFR alpha: 20 Units/50 ul.
Figure 2. Dose dependence curve of GST-PDGFR-alpha kinase activity: DELFIA® data generated using Phospho-Tyrosine mAb P-Tyr-100 #9411 to detect phosphorylation of substrate peptide (#1305) by PDGFR-alpha kinase. In a 50 ul reaction, increasing amounts of PDGFR-alpha and 1.5 uM substrate peptide were used per reaction well at 25ºC for 30 minutes. Background reading is 1624. (DELFIA® is a registered trademark of PerkinElmer, Inc.)
Figure 5. Staurosporine inhibition of PDGFR-alpha kinase activity: DELFIA® data generated using Phospho-Tyrosine mAb P-Tyr-100 #9411 to detect phosphorylation of PDGFR-alpha substrate peptide (#1305) by GST-PDGFR-alpha kinase. In a 50 ul reaction, 10 Units PDGFRalpha kinase, 1.5 uM substrate peptide, 20 uM ATP and increasing amounts of staurosporine were used per reaction well at 25ºC room temperature for 30 minutes. (DELFIA® is a registered trademark of PerkinElmer, Inc.)
Figure 3. Peptide concentration dependence of PDGFR-alpha kinase activity: DELFIA® data generated using Phospho-Tyrosine mAb P-Tyr-100 #9411 to detect phosphorylation of substrate peptide (#1305) by PDGFR-alpha kinase. In a 50 ul reaction, 10 Units of PDGFR-alpha and increasing concentrations of substrate peptide were used per reaction well at 25ºC for 30 minutes. Background reading is 597. (DELFIA® is a registered trademark of PerkinElmer, Inc.)
Figure 1. Time course of PDGFR-alpha kinase activity: DELFIA® data generated using Phospho-Tyrosine mAbP-Tyr-100 #9411 to detect phosphorylation of PDGFR-alpha substrate peptide (#1305) by PDGFR-alpha kinase. In a 50 ul reaction, 10 Units PDGFR alpha and 1.5 uM substrate peptide were used per reaction well. Background reading is 2421. (DELFIA® is a registered trademark of PerkinElmer, Inc.)