Figure 5. EGF-R 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:PolyEY, 10 ug/50 ul, Recombinant EGF-R: 5 Units/50 ul.
Figure 3. Dose dependence curve of EGF-R kinase activity: DELFIA® data generated using Phospho-Tyrosine Monoclonal Antibody PY100 #9411 to detect phosphorylation of peptide C03-1727 by GST-EGF-R kinase. In a 50 ul reaction, increasing amounts of GST-EGF-R and 5 uM substrate peptide were used per reaction well at 25ºC for 30 minutes. Background reading is 11152. (DELFIA® is a registered trademark of PerkinElmer, Inc.)
Figure 6. Iressa and staurosporine inhibition of EGF-R kinase activity: DELFIA® data generated using Phospho-Tyrosine Monoclonal Antibody P-Tyr-100 #9411 to detect phosphorylation of EGF-R substrate peptide (C03-1727) by GST-EGF-R kinase. In a 50 ul reaction, 10 Units GST-EGF-R , 1.5 uM substrate peptide, 20 uM ATP and increasing amount of inhibitor (iressa or staurosprine) were used per reaction well at 25ºC for 30 minutes. (DELFIA® is a registered trademark of PerkinElmer, Inc.)
Figure 4. Peptide concentration dependence of EGF-R kinase activity: DELFIA® data generated using Phospho-Tyrosine Monoclonal Antibody P-Tyr-100 #9411 to detect phosphorylation of peptide C03-1727 by GST-EGF-R kinase. In a 50 ul reaction, 10 Units of GST-EGF-R and increasing concentrations of substrate peptide were used per reaction well at 25ºC for 30 minutes. Background reading is 8985. (DELFIA® is a registered trademark of PerkinElmer, Inc.)
Figure 1. Time course of EGF-R kinase activity: DELFIA® data generated using Phospho-Tyrosine Monoclonal Antibody P-Tyr-100 #9411 to detect phosphorylation of EGF-R substrate peptide by GST-EGF-R kinase. In a 50 ul reaction, 10 Units GST-EGF-R and 5 uM substrate peptide were used per reaction well. Background reading is 5144. (DELFIA® is a registered trademark of PerkinElmer, Inc.).
Figure 2. Treatment of Tyrosine Kinase Substrate Screening Kit #7450 with EGF-R Kinase. Positive control is in yellow and negative control is in blue. Several positive hits for EGF-R were identified using this approach (in red). The star indicates the peptide chosen for this HTScan® EGF-R Kinase Assay Kit.