Figure 5. ABL1 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: PolyAEKY, 5 ug / 50 ul, Recombinant ABL1: 5 Units / 50 µl.
Figure 3. Dose dependence curve of ABL1 kinase activity: DELFIA® data generated using Phospho-Tyrosine Monoclonal Antibody P-Tyr-100 #9411 to detect phosphorylation of peptide C03-719 by GST-ABL1 kinase. In a 50 ul reaction, increasing amounts of GST-ABL1 and 1.5 uM substrate peptide were used per reaction well at 25ºC for 30 minutes. Background reading is 1481. (DELFIA® is a registered trademark of PerkinElmer, Inc.)
Figure 6. Staurosporine inhibition of ABL1 kinase activity: DELFIA® data generated using Phospho-Tyrosine Monoclonal Antibody P-Tyr-100 #9411 to detect phosphorylation of ABL1 substrate peptide (C03-719) by GST-ABL1 kinase. In a 50 ul reaction, 10 Units GST-ABL1 , 1.5 uM substrate peptide, 5 uM ATP and increasing amount of staurosporine were used per reaction well at 25ºC for 30 minutes. (DELFIA® is a registered trademark of PerkinElmer, Inc.).
Figure 4. Peptide concentration dependence of ABL1 kinase activity: DELFIA® data generated using Phospho-Tyrosine Monoclonal Antibody P-Tyr-100 #9411 to detect phosphorylation of peptide C03-719 by GST-ABL1 kinase. In a 50 ul reaction, 10 Units of GST-ABL1 and increasing concentrations of substrate peptide were used per reaction well at 25ºC for 30 minutes. Background reading is 871. (DELFIA® is a registered trademark of PerkinElmer, Inc.)
Figure 1. Time course of ABL1 kinase activity: DELFIA® data generated using Phospho-Tyrosine Monoclonal Antibody P-Tyr-100 #9411 to detect phosphorylation of ABL1 substrate peptide by GST-ABL1 kinase. In a 50 ul reaction, 10 Units GST-ABL1 and 1.5 uM substrate peptide were used per reaction well. Background reading is 768. (DELFIA® is a registered trademark of PerkinElmer, Inc.)
Figure 2. Treatment of Tyrosine Kinase Substrate Screning Kit #7450 with GST-ABL1 Kinase. Positive control is in yellow and negative control is in blue. Several positive hits for ABL1 were identified using this approach (in red). The star indicates the peptide chosen for this HTScan® ABL1 Kinase Assay Kit.